Mycotoxin Binder

ABSTRACT

A mycotoxin binder is disclosed characterized by 45% or more humic acid, maximum solubility of 20% at pH between 1.5 and 7.0, and an in vitro mycotoxin binding efficiency of at least 80% and preferably 90% with adsorption of at least 85% at pH 3.0 and desorption less than 10% at pH 6.8.

This application claims priority to U.S. patent application Ser. No.13/109,569, filed May 17, 2011, which claims priority to U.S. PatentApplication Ser. No. 61/345,186 filed May 17, 2010 and incorporates thesame herein in their entireties by this reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to mycotoxin binders and, morespecifically, to mycotoxin binders utilizing humic compounds.

Mycotoxins are invisible, odorless and cannot be detected by smell ortaste, but can result in great economic losses at all levels ofagricultural feed production and especially in animal production.Mycotoxins are secondary metabolites produced by filamentous fungi suchas Fusarium, Aspergillus, and Penicillium prior to and during harvest,or during (improper) storage. Their toxic effects are very diverse(Akande, K. E., Abubakar, M. M., Adegbola, T. A., and Bogoro, S. E. 2006Nutritional and Health Implications of Mycotoxins in Animal Feeds: AReview. Pakistan Journal of Nutrition, 5: 398-403). In farm animals,mycotoxins have negative effects on feed intake, animal performance,reproductive rate, growth efficiency, immunological defense as well asbeen carcinogenic, mutagenic, teratogenic, cause tremors or damage thecentral nervous system, hemorrhagic, as well as causing damage to theliver and kidneys. Mycotoxins are metabolized in the liver and thekidneys and also by microorganisms in the digestive tract. Therefore,often the chemical structure and associated toxicity of mycotoxinresidues excreted by animals or found in their tissues are differentfrom the parent molecule (Ratcliff, J. The Role of Mycotoxins in Foodand Feed Safety. Presented at Animal Feed Manufacturers Association,Aug. 16, 2002). Various mycotoxins may occur simultaneously, dependingon the environmental and substrate conditions (Sohn, H. B., Seo, J. A.,and Lee, Y. W. 1999 Co-occurrence of Fusarium Mycotoxins in Mouldy andHealthy Corn from Korea. Food Additives and Contaminants, 16: 153-158).Considering this coincident production, it is very likely, that animalsare exposed to mixtures rather than to individual compounds. Fieldstudies have shown that more severe toxicosis in animals can result fromthe additive and synergistic effects of different mycotoxins (Ratcliff,2002). The problem of mycotoxins does not just end in animal feed orreduced animal performance, many become concentrated in meat, eggs andmilk of animal and can pose a threat to human health. There isincreasing concern about levels of mycotoxins in human foods, both fromvegetable origin and animal origin.

Although there are geographic and climatic differences in the productionand occurrence of mycotoxins, exposure to these substances is worldwide.Mycotoxins are estimated to affect as much as 25 percent of the world'scrops each year (Akande, 2006). Most countries have stringent regulationon mycotoxin levels in feed and the main goal of agricultural and foodindustries is the prevention of mycotoxin contamination in the field.Management practices to maximize plant performance and decrease plantstress can decrease mycotoxin contamination substantially. This includesplanting adapted varieties, proper fertilization, weed control,necessary irrigation, and proper crop rotation (Edwards, S. G. 2004Influence of Agricultural Practices on Fusarium Infection of Cereals andSubsequesnt Contamination of Grain by Tricothecenes Mycotoxins.Toxicology Letters, 153: 29-35). But even the best management strategiescannot eliminate mycotoxin contamination in years favorable for diseasedevelopment.

Among the various mycotoxins identified especially affecting poultry,some occur significantly in naturally contaminated foods and feeds. Theyare aflatoxin; ochratoxin, zearalenone, T-2 toxin, vomitoxin andfumonisin. They cause detrimental effects on birds, such as growthimpairment, immune depression, and paleness in broilers, which finallybring out economic losses.

Aflatoxin B1, a metabolite of fungus Aspergillus flavus and Aspergillusparasiticus, is an extremely hepatotoxic compound that frequentlycontaminates poultry feeds at low levels. Another family of mycotoxinsproduced by Penicillium and Aspergillus genera is ochratoxin.Ochratoxin, being the most potent toxin, adversely affects productionparameters and the health of poultry. Ingestion of ochratoxin causessevere kidney damage. T-2 toxin induces severe inflammatory reactionsand neural disturbances in animals and humans, whereas zearalenoneappears to have no effect on poultry health and performance. Poultryrations with high levels of Fusarium contamination have been associatedwith poor performance, feed refusal, diarrhea, leg weakness, orallesions, and/or high mortality.

The toxicity and clinical signs observed in animals when more than onemycotoxin is present in feed are complex and diverse. Mycotoxins areusually accompanied by other unknown metabolites which may havesynergistic or additive effects. The ability of binders to alleviate theadverse effects of the several combinations of mycotoxins presentnaturally in feed on productivity and serum biochemical andhematological parameters remains yet to be explored.

Practical methods to detoxify mycotoxin contaminated grain on a largescale and in a cost-effective manner are not currently available. Atpresent, one of the more promising and practical approaches is the useof adsorbents. However, several adsorbents have been shown to impairnutrient utilization (Kubena, L. F., R. B. Harvey, T. D. Phillips, D. E.Corrier, and W. E. Huff. Diminution of aflatoxicosis in growing chickensby the dietary addition of hydrated sodium calcium aluminosilicate.Poult. Sci. 69:727-735. 1990) and mineral adsorption (Chestnut, A. B.,P. D. Anderson, M. A. Cochran, H. A. Fribourg, and K. D. Twinn. 1992.Effects of hydrated sodium calcium aluminosilicate on fescue toxicosisand mineral absorption. J. Anim. Sci. 70:2838-2846) and lack bindingeffects against multiple mycotoxins of practical importance (Edrington,T. S.; Sarr, A. B.; Kubena, L. F.; Harvey, R. B.; Phillips, T. D.(1996). Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS,and activated charcoal reduce urinary excretion of aflatoxin M1 inturkey poults. Lack of effect by activated charcoal on aflatoxicosis.Toxicology letter, 89: 115-122).

Zearalenone (ZEA) causes hyperestrogenism in swine when ingested atlevels as low as 1 μg/g feed. Pathology in swine is more pronounced inprepubertal gilts and are characterized by tumefaction of the vulva,prolapses of the vagina and rectum and enlargement of the mammaryglands. In cycling animals, effects of zearalenone include conceptionfailure, pseudopregnancy and abortion. The metabolism of ZEA seems tooccur essentially in the liver leading to α and β zeatalenol. The enzymebelieved to catalyze reduction of ZEA to zearalenol is3-α-hydroxysteroid dehydrogenase (3α-HSD). This enzyme is also known todegrade 5-α androstan-3,17-dione, a product of steroid hormonemetabolism. As known in several studies, ZEA and its metabolites areexcreted mainly via feces and urine. Swine are more sensitive to ZEAthan other classes of livestock, and feeding regimens that minimizelosses due to feed wastage and poor performance are desirable. Somecompounds (i.e. fiber, formalin, sodium carbonate and monomethylamine)has been shown to protect against numerous xenobiotisc, including ZEAeffects.

The use of mold inhibitors or preservation by acids can only reduce theamount of mold but does not influence the content of mycotoxinsgenerated prior to treatment. If mycotoxins have been produced earlierthey will not be affected in any form by mold inhibitors or acidmixtures, as they are very stable compounds. Thus these toxic compoundsremain in the formerly infected commodity even if no further mold can beseen or detected. The most commonly used strategy of reducing exposureto mycotoxins is the decrease in their bioavailability by the inclusionof various mycotoxin binding agents or adsorbents, which leads to areduction of mycotoxin uptake and distribution to the blood and targetorgans. Major advantages of adsorbents include expense, safety and theease to add to animal feeds. Various substance groups have been testedand used for this purpose, with aluminum silicates, in particular clayand zeolitic minerals, as the most commonly applied groups.

Humic acids are ubiquitous and are found wherever matter is beingdecomposed or has been transposed, as in the case of sediments. They arenatural components of drinking water, soil and lignite. Humic substanceshave a strong affinity to bind various substances, such as heavy metals,herbicides, different mutagens, monoaromatic and polycyclic aromaticcompounds and minerals. Farmers use humates to accelerate seedgermination and improve rhizome growth for many years (Islam, K. M.,Schuhmacher, S. A., and Gropp, M. J. 2005 Humic Substances in AnimalCulture. Pakistan Journal of Nutrition, 4: 126-134). The materials areable to stimulate oxygen transport, accelerate respiration and promoteefficient utilization of nutrient by plants (Osterberg, R. andMortensen, K. 1994 The Growth of Fractal Humic Acids: ClusterCorrelation and Gel Formation. Radiation and Environmental Biophysics,33: 269-276). These observations prompted scientists to study thespecific properties of humates and their possible benefits in improvinghealth and well being of humans and animals. Humic substances have beenused as an anti-diarrheal, analgesic, immune-stimulatory and growthpromoting agents in veterinary practices in Europe (Islam, 2005). Humicacids inhibit bacterial and fungal growth, thus indirectly decreaselevels of mycotoxins in feed (Riede, U. N., Zeck-Keapp, G., Freudenberg,N., Keller, H. U., and Seubert, B. 2007 Humate Induced Activation ofHuman Granulocytes. Virchows Archives of Biology: Cell Pathology, 60:27-34). Some humic substances and their salts have been described todirectly interact with mycotoxins by their mycotoxin binding capacity(Sabater-Vilar, M., Malekinejad, H., Selman, M. H. J., Ven Der Doelen,M. A. M., and Fink-Gremmels, J. 2007, In Vitro Assessment of AbsorbentsAiming to Prevent Deoxynivalenol and Zearalenone Mycotoxicosis.Micropathologia, 163: 81-90; Ye, S., Lv, X., and Zhou, A. 2009 In VitroEvaluation of the Efficacy of Sodium Humate as an Aflatoxin B1Adsorbent. Australian Journal of Basic and Applied Sciences, 3:1296-1300; Jansen van Rensburg, C., Van Rensburg, C. E. J., Van Ryssen,J. B. J., Casey, N. H., and Rottinghaus, G. E. 2006 In Vitro and In VivoAssessment of Humic Acid as an Aflatoxin Binder in Broiler Chickens.Poultry Science, 85: 1576-1583).

SUMMARY OF THE INVENTION

The new group of mycotoxin binders disclosed are humic acid containingsubstances. Preferably, the mycotoxin binders have a minimum humic acidcontent of 45%, maximum solubility of approximately 20% at pH 1.5, 3.0and 7.0, and in vitro mycotoxin binding efficiency of at least 80%, andpreferably 90%, with minimal adsorption at the pH of the stomach of amonogastric animal of at least 80%, and preferably at least 85%, andmaximal desorption at neutral pH of not greater than 10%. In a preferredembodiment, the humic substances are combined with an adsorbent, such asclay, to provide an effective mycotoxin binder in vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart of average weekly vulva scores for all groups ofgilts; standard errors ranged from 0.3 to 0.5.

FIG. 2 is a chart of the evolution of average estradiol-17β plasmaconcentration for different batches.

FIG. 3 is a chart of the percentage of ovaries having follicules ≦6 mmin size for different batches.

FIG. 4 is a chart of the percentage of ovaries having follicules >6 mmin size for different batches.

FIG. 5 is a chart of the percentage of gilts having hypoplasic ovary andfollicules >6 mm in size.

DESCRIPTION OF THE INVENTION

The present invention includes compositions to be added to animal feedsthat may be contaminated with one or more mycotoxins. The compositionsinclude humic substances, preferably humic substances containing between45% and 99% humic acids, including all levels within such range. Thecompositions should have a high affinity for the specific mycotoxinsbeing addressed, resulting in the formation of a strong complex betweenthe composition and the targeted mycotoxin that will not be disrupted bysolubility in the gastrointestinal tract so that the mycotoxin will beexcreted in the feces. Accordingly, it is preferred that thecompositions not have solubility greater than 20% at pH between 1.5 to7.5. Compositions of the present invention found to be effective in vivohave in vitro binding efficiencies of greater than 80%, and preferablygreater than 90%, with minimal absorption at ph 3.0 of at least 80%, andpreferably 85%, and maximal desorption at pH 6.8 of not more than 10%.

Humic substances suitable for the present invention can be obtained frommany sources, but a preferred source is leonardite. The humic substancesare preferably combined with one or more sources of metal ions, oxidesand clay minerals. Preferably, the humic substances are combined with aclay, such as bentonite or sepiolite, in amounts ranging from 10% to 90%of a source of humic substances combined with amounts of the clayranging from 90% to 10%, and all ratios in between.

Humic Acids in Natural Substances

Humic acids are formed through the chemical and biological humificationof organic matter, particularly plants and through the biologicalactivities of micro-organisms. They are found in the brown organicmatter of a variety of soils, as well as in peats, manure, lignite,leonardite and brown coals. In soils they also may be formed by certainsecondary processes such as polymerization of polyphenols leached byrain from surface leaf litter, and condensation of phenols, quinones,and proteins that are provided by the action of soil micro-organisms andsmall animals on soil carbohydrates.

Humic acids do not have a single unique structure, but are a mixture ofintermediate chemical products resulting from the decomposition andconversion of lignin and other plant materials to hard coal. They arethree dimensional macrocolloidal molecules with a polyaromatic centercontaining iso- and heterocyclic structures and peripheral side-chains.The organic structure of humic acid is naturally oxidized, giving it anegative charge. Positive ions, attracted to broken bonds at the site ofthe oxidation, create sites for micronutrients and micro-flora toattach. Low-grade coals, called lignite, contain more acids thanhigh-grade coals. Leonardite is a particular formation of highlyoxidized lignite. This material has the highest humic acids content ofany natural source.

Example 1

An experiment was conducted to determine the humic acid content of fivenatural humic acid containing substances. The humic acids were measuredby a volumetric method with titration of the extracts according to theInternational Standard (ISO 5073:1999 Brown coals and lignites.Determination of humic acids).

The results of the analyses of the humic acid containing substances areshown in Table 1. The disclosed minimum requirement of 45% humic acidwas exceeded by all products, except for HS3 (28.34%) and HS4 (44.62%).

TABLE 1 Humic acid content determined by ISO 5073: 1999. Humic acidcontaining substance Humic acid content (%) HS1 46.31 HS2 64.84 HS328.34 HS4 44.62 HS5 67.45

Solubility of Humic Acid Containing Substances at Different pH

Most humic acid substances are chemically attached to inorganiccomponents (clay or oxides), and a smaller part gets dissolved in thesolutions or the soil, particularly under alkaline conditions. Animportant feature of humic substances is that they can combine withmetal ions, oxides and clay minerals to form water soluble or insolublecomplexes and can interact with organic compounds such as alkenes, fattyacids, capillary-active substances and pesticides.

Adsorbents used to hinder the gastrointestinal absorption of mycotoxinsshould have a high affinity for the specific mycotoxins, resulting inthe formation of a strong complex that will not be disrupted and that isexcreted via the feces. This implicates that humic acids, as the mainactive compounds, may not be dissolved at any location in thegastrointestinal tract. The disclosed humic acid substances may not havesolubility higher than 20% at pH 1.5; 3.0 and 7.0 (i.e. minimal 80%retentate recovery).

Example 2

An experiment was conducted to determine the non-soluble part of humicacid containing substances. Briefly, an amount of 0.15 g product wasdissolved in 75 ml 0.1M phosphate buffer (adjusted to pH 1.5; 3.0 or7.0) and was incubated for one hour at room temperature on a magneticstir plate (600 rpm). All suspensions were filtered through a 55 mmfilter (Macherey-Nagel, MN GF-4). The retentate was dried for two hoursat 130° C. The retentate amount was calculated as the mass differencebetween empty filter and filter+dried retentate. Recovery (%) wascalculated as the ratio ‘retentate amount’/‘mass product’ and expressedas percentage. The samples were analyzed in triplicate and means werecalculated. The reference material was purified humic acid (HA, SigmaAldrich. Lot 0001411101, cas number: 1415-93-6).

Recovery of the non-soluble fraction of the HA suspension at pH 7 wasnot possible. Filtration of this suspension could not be performed,because of ‘floating substances’ that were formed during the incubationstep which made the filter non-permeable. The experiment was repeated incitrate buffer adjusted to pH 6.2. However the results were the same.The other suspensions of the products could easily be filtered at eachpH tested. The results of the recovered retentates are shown in Table 2.All but two products met the minimum retentate recovery of 80% at thedifferent pH conditions. Recovery of the retentate of HS 1 was too lowat pH 1.5; recovery of the retentates of HS3 was too low at all pHlevels tested.

TABLE 2 Non-soluble fraction of humic acid containing substances atdifferent pH, expressed as % mass recovered in retentate. pH 1.5 3 7mean stdev mean stdev mean stdev HA 80.97 4.13 84.65 3.18 ND ND HS178.41 0.97 80.46 1.14 85.40 0.48 HS2 85.02 1.21 86.54 0.80 84.23 0.75HS3 67.80 0.78 68.16 0.93 64.14 2.62 HS4 80.75 1.58 84.54 2.30 92.130.42 HS5 83.24 0.85 84.73 0.46 86.47 2.02 ND: retenate recovery of HA atpH 7 not determined

Example 3

In a second experiment on solubility of humic acid containingsubstances, 0.015 g (instead of 0.15 g) of the products HS2, HS3 and HAwas dissolved in 75 ml 0.1M phosphate buffer (pH 1.5; 3.0 or 7.0).

After incubation for one hour at room temperature on a magnetic stirplate, the morphology of the suspensions of the three products testedclearly differed (FIG. 1). The results of the recovered retentates areshown in Table 3. The dark brown color of the suspension of HA at pH 7corresponded with high solubility of the product at this pH (Table 3).

TABLE 3 Non-soluble fraction of humic acid containing substances atdifferent pH, expressed as % mass recovered in retentate. pH 1.5 3 7mean stdev mean stdev mean stdev HA 103.17 2.69 128.41 8.93 54.82 5.74HS2 107.86 10.44 115.98 7.56 121.25 11.06 HS3 106.88 8.59 126.59 9.5791.24 2.02

In Vitro Mycotoxin Binding Capacity of Humic Acid Containing Substances

During the adsorption process the mycotoxin is not really bound to thesurface of the binder. The electrostatic forces that join the toxin withthe binder are not permanent links, which means that the adsorptionprocess is reversible. A change of the surrounding environment of themycotoxin binder (e.g. in the digestive tract) can have a dramaticeffect on the binding efficacy. The major parameter that has aninfluence is the pH of the environment. Changes in pH can change boththe mycotoxin and the surface of the binder, causing a modification ofthe attraction between the two. In an animal, in the foregut theconditions of low pH may promote adsorption of mycotoxins, while furtherin the digestive tract (more neutral pH) the mycotoxin may be releasedagain. Because of the vast effect of pH on the adsorption it is ofprimordial importance to use an in vitro system that mimics the changeof the pH conditions along the gastrointestinal tract.

Example 4

A study was performed on the in vitro evaluation of the mycotoxindetoxifying efficiency of potential sequestering humic acid containingsubstances against zearalenone. Adsorption in an acidic environment (pH3.0)—mimicking the pH of the stomach of a monogastric animal—anddesorption at near neutral pH (pH 6.8)—mimicking pH conditions in theintestine of a monogastric animal—were measured. The net percentage ofmycotoxin detoxifying efficiency was determined as adsorption percentageminus desorption percentage.

Duplicate aliquots of 0.1M phosphate buffer (adjusted to pH 3.0)containing 300 ppb zearalenone in solution (10 ml) were added to 15 mlscrew cap Falcon polypropylene tubes to which had been added 0.05 gramof each adsorbent. Test tubes were placed on an orbital shaker for 60minutes at room temperature. Each mycotoxin test solution wascentrifuged at 4000 rpm for 10 minutes. The aqueous supernatant wasisolated for mycotoxin analysis (adsorption). The pellet was resuspendedin 0.1 M phosphate buffer pH 6.8. Test tubes were placed again on anorbital shaker for 60 minutes at room temperature and afterwardscentrifuged at 4000 rpm for 10 minutes. The aqueous supernatant wasanalyzed for zearalenone (desorption). The zearalenone concentrationswere determined using ELISA (Euro-Diagnostica). Buffered zearalenonetest solutions (pH 3.0 or 6.8) without adsorbents were used as standard.

A summary of the in vitro mycotoxin adsorption by five samples ispresented in Table 4. The results showed that all the productseffectively adsorbed zearalenone at pH 3.0 (adsorption above the minimalrequired value of 85%), except for HS3. Notable differences wereobserved for the desorption at pH 6.8. The products HS3 and HS4 bothwere shown to exceed the maximal value of 10% desorption. The otherthree samples met the requirement. Only products HS2 and HS5 fulfilledthe minimal required in vitro binding efficacy of 90%.

TABLE 4 In vitro binding of zearalenone by humic acid containingsubstances at pH 3.0 and 6.8. in vitro ZEA Binding Absorption (%)Desorption (%) Efficiency (%) Adsorbent mean SD mean SD mean HS1 92.325.01 8.91 0.48 83.40 HS2 96.20 2.39 3.12 1.77 93.08 HS3 80.68 35.7044.99 HS4 86.65 13.54 26.57 0.40 59.95 HS5 96.78 2.07 1.70 0.28 96.36

Example 5

Two humic acid substances (HS2 and HS3) were compared to purified humicacid (HA, Sigma Aldrich. Lot 0001411101, cas number: 1415-93-6) in an invitro mycotoxin binding assay similar to the test described in example4, with the only modification that the zearalenone detection wasperformed by HPLC analysis, after extraction of the mycotoxin by an AOZcolumn (VICAM, USA).

The HPLC analyses were performed on an SP8800 Ternary LC SolventDelivery System with helium degassing (Spectra Physics, USA), a SP 8880autosampler (Spectra Physics, USA) with 20 μl loop, a Chromjetintegrator (Thermo, USA), a Croco Cil™ column heater (Cluzeau Info Labo,France) and an UV-fluorescence detector FP-920 (Jasco, Japan), ChromsepNucleosil 100-5 C18 SS 250*4.6 mm (L*ID) columns (Varian, theNetherlands) or equivalent. The columns were protected with anappropriate guard column. An aliquot of the original bufferedzearalenone test solution was used as the HPLC standard.

The results are summarized in Table 5. The data of HS2 and HS3 confirmedthe results of example 4. Adsorption of zearalenone on purified humicacid at pH 6.8 was very high (96.93%). However at pH 6.8, 30.83% of thebounded zearalenone was released again.

TABLE 5 In vitro binding of zearalenone by humic acid containingsubstances at pH 3.0 and 6.8. Results are mean values of duplicateanalysis. For HS3, only single analyses were available. in vitro ZEABinding Absorption (%) Desorption (%) Adsorbent mean SD mean SDEfficiency (%) HS2 94.40 0.56 2.96 0.48 91.44 HS3 86.03 31.71 54.33 HA96.93 0.36 30.83 0.51 66.10

In conclusion, two products HS2 and HS5 fulfilled all three preferredcharacteristics for improved mycotoxin binders of the present invention.

Example 6 Materials and Methods

Solutions Used for Total Humic Acid Determination in Humic AcidContaining Substances.

Humic acids were measured by a volumetric method with titration of theextracts according to the International Standard (ISO 5073:1999 Browncoals and lignites. Determination of humic acids). Table 6 gives anoverview of the different solutions used in the procedure.

TABLE 6 Solutions used for humic acid determination Solution CompositionSodium 15 g Na₄P₂O₇(10H₂O) + 7 g NaOH in H₂O pyrophosphate up to 1 LSodium hydroxide 10 g NaOH in H₂O up to 1 L Potassium dichromate 4.9036g K₂Cr₂O₇, previously dried at 130° C., standard solution in H₂O up to 1L Potassium dichromate 20 g K₂Cr₂O₇ in H₂O up to 1 L oxidizing solution1,10-phenanthroline 1.5 g 1,10-phenanthroline + 1 g indicator(NH₄)2Fe(SO₄)2•6H₂O Sulphuric acid concentrated, ρ₂₀ = 1.84 g/mlAmmonium ferrous 40 g (NH₄)2Fe(SO₄)2•6H₂O + 20 ml H₂SO₄ sulphate in H₂Oup to 1 L

The ammonium ferrous sulphate titration solution was standardizedagainst the potassium dichromate standard solution for each batch ofsamples to be analyzed. Twenty five ml of potassium dichromate standardsolution was pipetted into a 300 ml conical flask. Seventy ml was addedto 80 ml of water. Carefully 10 ml of concentrated sulphuric acid and 3drops of 1,10-phenantholine indicator were added. After cooling, thesolution was titrated with the ammonium ferrous sulphate standardsolution to a red colour. The concentration was calculated, in moles perL, of the ammonium ferrous sulphate solution as follows (1): where c isthe concentration, expressed in moles per L, of the ammonium ferroussulphate solution; V is the volume of ammonium ferrous sulphate solutionrequired for the titration.

$\begin{matrix}{c = {0.1 \times \frac{25}{V}}} & (1)\end{matrix}$

Extraction of Total Humic Acids.

Five humic acid containing substances, hereafter called leonardite, wereobtained. Table 7 gives an overview of the source of each leonardite.For the extraction of the humic acids, 0.2±0.0002 g of analysis samplewas weighed into a conical flask. Hundred fifty ml of alkaline sodiumpyrophosphate extraction solution (total humic acids) was added andmixed until the sample was thoroughly wetted. A small funnel was placedon the flask and heated in the boiling water bath for 2 h, shakingfrequently to ensure precipitation of insoluble material. The flask wasremoved from the water bath, allowed to cool to room temperature and theextract and residue was transferred to a 200 ml volumetric flask. Theextract was diluted to the mark with water and shaken to ensure thoroughmixing.

Determination of Total Humic Acids.

For the determination of the humic acids in the extracts, 5 ml ofextract was pipetted into a 250 ml to 300 ml conical flask. Five ml ofpotassium dichromate oxidizing solution was added into the flask.Carefully 15 ml of concentrated sulphuric acid was added. The solutionwas placed in a boiling water bath for 30 min. the solution was cooledto room temperature and diluted to approximately 100 ml. Three drops of1,10-phenanthroline indicator were added to the solution and titratedwith the ammonium ferrous sulfate titration solution to a brick redcolor. As blank samples for total humic acids, 5 ml of extract isreplaced by 5 ml sodium pyrophosphate.

Calculation of Total Humic Acids.

The total humic acid content (w_(HA,t)) was calculated as a percentageby mass, of the sample as analysed according to the following formula(2) where 0.003 is the millimole mass of carbon, in g/mmol; V₀ is thevolume of the ammonium ferrous sulphate titration solution used in theblank titre, in ml; V₁ is the volume of the ammonium ferrous sulphatetitration solution used in the extract titre, in ml; c is theconcentration of the ammonium ferrous sulphate titration solution, inmol/L; V_(e) is the volume of the extract, in ml; V_(a) is the volume ofthe aliquot taken for titration, in ml; 0.59 is the average ratio ofcarbon content of humic acids for brown coals and lignites; m is themass of the sample taken for the test, in g.

$w_{HA} = {\frac{\left( {V_{0} - V_{1}} \right) \times 0.003 \times c}{0.59 \times m} \times \frac{V_{e}}{V_{a}} \times 100}$

TABLE 7 Sources of leonardite Leonardite Source HS1 Xuguang Jieneng Co.,Ltd, Yunnan Province, China HS2 Poortershaven, Rotterdam, TheNetherlands HS3 HuminTech, Düsseldorf, Germany HS4 Pingxiang JialiCeramic Materials Co., Ltd, Jiangxi Province, China HS5 Double DragonsHumic Acid Co. Ltd. Xinjiang, China

Solubility of Leonardite Samples at Different pH.

An experiment was conducted to determine the non-soluble part ofleonardite samples. Briefly, 0.15 g of sample was dissolved in 75 mlsolution with adjusted pH of 1.5, 3.0 or 7.0. The solution at pH 1.5 wasa Clark and Lubs solution composed of 25 ml 0.2M KCl, 20.7 ml 0.2M HCldiluted to 100 ml with MilliQ water (Millipore, Brussels, Belgium). Thesolution at pH 3.0 was 0.1M NaH₂PO₄ adjusted to pH using H₃PO₄ (Acros,Geel, Belgium); the solution at pH 7.0 was 0.1M Na₂HPO₄.2H₂O adjusted topH using 0.1M NaH₂PO₄. All chemicals were from VWR International,Leuven, Belgium). The suspension was incubated for one hour at roomtemperature on a magnetic stir plate (600 rpm). After one hour, allsuspensions were filtered through a 55 mm glass fibre filter (GF-92,Whatman, Dassel, Germany). The retentate was dried for two hours in anoven at 130° C. The retentate amount was calculated as the massdifference between empty filter and filter+dried retentate. Recovery (%)was calculated as the ratio ‘retentate amount’/‘mass product’ andexpressed as percentage. The samples were analysed in triplicate andmeans were calculated. The reference material was purified humic acid(HA, Sigma Aldrich, Bornem, Belgium). Recovery of the non-solublefraction of the HA suspension at pH 7 was not possible. Filtration ofthis suspension could not be performed, because of ‘floating substances’that were formed during the incubation step which made the filternon-permeable. The experiment was repeated in 0.1M citrate solution(C₆H₅Na₃O₇.2H₂O in MilliQ water) adjusted to pH 6.2 using C₆H₈O₇.H₂O forsamples HS2, HS3 and HA. However the results were the same. The othersuspensions of the products could easily be filtered at each pH tested.In order to resolve the solubility issue of humic acid at pH 7, theexperiment was repeated using only 0.02 g of sample instead of 0.15 g.Only samples HS2, HS3 and HA were repeated. All other test conditionsremained the same.

In Vitro Mycotoxin Binding Capacity of Humic Acid Containing Substances.

Details about the in vitro method can be found in the internalinstruction LB-IV-20/142-E²; Determination of mycotoxin detoxifyingefficiency in a “two-phase procedure” coupled with HPLC analysis.However, modification of this procedure was necessary because theleonardite included in the new toxin binder product severely interferedwith HPLC detection of the mycotoxins when supernatants were injecteddirectly in the HPLC system. Therefore, an immunoaffinity column (IAC,Vicam, USA) clean-up step is used (see below for more details).

Adsorption in an acidic environment (pH 3.0)—mimicking the pH of thestomach of a monogastric animal—and desorption at near neutral pH (pH6.8)—mimicking pH conditions in the intestine of a monogastricanimal—were measured. The net percentage of mycotoxin detoxifyingefficiency was determined as adsorption percentage minus desorptionpercentage. Duplicate aliquots of 0.1M phosphate solution (adjusted topH 3.0) containing 300 ppb zearalenone in solution (10 ml) were added to15 ml screw cap Falcon polypropylene tubes to which had been added 0.05gram of each adsorbent. Test tubes were placed on an orbital shaker for60 minutes at room temperature. Each mycotoxin test solution wascentrifuged (5000-12000 g) for 10 minutes until a clear solution wasobtained. The aqueous supernatant was isolated for zearalenone analysis(adsorption). The pellet was resuspended in 0.1M phosphate solution pH6.8. Test tubes were placed again on an orbital shaker for 60 minutes atroom temperature and afterwards centrifuged. The aqueous supernatant wasisolated for zearalenone analysis (desorption). After centrifugation ofthe solution at pH 3.0 and 6.8, an IAC clean-up step is introduced.After discarding the protective liquid from the IAC columns, columnswere washed with 8 ml phosphate buffered saline (PBS) solution pH 7.4.Then, a 3 ml of supernatant was applied to the column. Afterwards, thecolumn was washed with 20 ml mQ water and dried using mild vacuum for afew seconds. Zearalenone was eluted with 3 ml of 2% (v/v) glacial aceticacid in methanol (MeOH) into glass tubes. The whole eluate wasevaporated until dry under a gentle stream of N₂ at 60° C. andredissolved in 1.5 ml acetonitril/mQ water 60/40 (v/v) prior to HPLCanalysis. The zearalenone concentrations were determined using both HPLC(all samples) and ELISA (HS1, HS2, HS4, HS5). Zearalenone test solutions(pH 3.0 or 6.8) without adsorbents were used as standard.

Results

Determination of Total Humic Acid Content in Different LeonarditeSamples.

The results of the analyses are shown in Table 8. The total humic acidcontent in samples 2 and 5 is the highest; sample 1 and 4 show mediumlevels and sample 3 is the lowest.

TABLE 8 Total humic acid content in leonardite samples (n = 2) SampleAverage SD HS1 46.31 0.81 HS2 64.84 0.29 HS3 31.01 0.38 HS4 44.62 0.36HS5 67.45 2.15

Solubility of Leonardite Samples at Different pH.

The results of the recovered retentates at different pH using aphosphate solution are shown in Table 9. Sample HS3 showed the highestsolubility over the tested pH range. The retentate of humic acid at pH 7could not be recovered. At pH 7 it was completely dissolved, formingslurry. This slurry blocked the filters. The test was repeated with HA,HS2, HS3 and a citrate solution at pH 6.2; Table 10 shows the resultsfor HA were the same using the citrate solution. The results of therecovered retentates using only 0.02 g of HA, HS2, HS3 and the phosphatesolution are shown in Table 11. Because of the lower sampleconcentration the filters did not block and recovery could becalculated. It is clear that humic acid shows the highest solubility atpH 7 compared to all other samples. This was also confirmed by visualinspection of the samples after incubation. Due to the error in weighingand recovering small sample quantities, the standard deviations werelarge. Therefore preference should be given to using 0.2 g of sample.

TABLE 9 Non-soluble fraction of leonardite, expressed as % massrecovered in retentate (phosphate, n = 3). pH 1.5 3 7 mean stdev meanstdev mean stdev HA 80.97 4.13 84.65 3.18 / / HS1 78.41 0.97 80.46 1.1485.40 0.48 HS2 85.02 1.21 86.54 0.80 84.23 0.75 HS3 67.80 0.78 68.160.93 64.14 2.62 HS4 80.75 1.58 84.54 2.30 92.13 0.42 HS5 83.24 0.8584.73 0.46 86.47 2.02

TABLE 10 Non-soluble fraction of leonardite, expressed as % massrecovered in retentate (citrate, n = 3). mean stdev HA / / HS2 100.822.20 HS3  53.06 1.96

TABLE 11 Non-soluble fraction of leonardite, expressed as % massrecovered in retentate (0.02 g sample, n = 3). pH 1.5 3 7 mean stdevmean stdev mean stdev HA 103.17 2.69 128.41 8.93 54.82 5.74 HS2 107.8610.44 115.98 7.56 121.25 11.06 HS3 106.88 8.59 126.59 9.57 91.24 2.02

In Vitro Mycotoxin Binding Capacity of Leonardite.

A summary of the in vitro mycotoxin adsorption analysed by ELISA ispresented in Table 12; by HPLC in Table 13. The results show that allthe products effectively adsorb zearalenone at pH 3.0, HS3 having thelowest adsorption. Notable differences were observed for the desorptionat pH 6.8. HS3 and HS4 both show a high degree of desorption compared tothe other samples.

TABLE 12 In vitro binding of zearalenone by leonardite samples, assessedusing ELISA (n = 4). Adsorption(%) Desorption(%) Efficacy(%) mean stdevmean stdev mean stdev HS1 92.32 5.01 8.91 0.48 83.40 4.53 HS2 96.20 2.393.12 1.77 93.08 4.16 HS4 86.65 13.54 26.57 0.40 59.95 14.00 HS5 96.782.07 1.70 0.28 96.36 2.04

TABLE 13 In vitro binding of zearalenone by leonardite samples, assessedusing HPLC (n = 3). Adsorption (%) Desorption (%) Efficacy (%) meanstdev mean stdev mean stdev HA 96.93 0.36 30.83 0.51 66.10 0.19 HS294.40* 0.56 2.96 0.48 91.44 1.04 HS3 82.47 3.13 34.37 3.74 48.10 6.39 *n = 2

Example 7 Materials and Methods

Experimental Design.

60 female weaned piglets (Rongchang×Dabai crossbred) with an averageweight of 9 kg were randomly assigned to 5 different groups. There were3 replicates for each group with 4 piglets per replicate. The fivegroups were: 1) negative control (NC), 2) positive control (PC), 3)Toxfin Supreme (TS)1, 4) TS2 and 5) TS3. The NC group was fed clean (noZEA contamination) basal diets without Toxfin Supreme; the PC group wasfed ZEA contaminated diets (1 ppm) without Toxfin Supreme; and TS 1, TS2and TS3 groups were fed ZEA contaminated diets (1 ppm) supplemented with1, 2 and 3 g/kg Toxfin Supreme, respectively. All piglets were reared infloor pens and ad libitum access to feed and water. All piglets were fedbasal diets for one week prior the start of the trial, so that theycould adapt to the experimental environment.

Zearalenone Contaminated Feed Preparation.

The nutrient levels of the basal diets met NRC requirements (Table 14).About 800 kg of corn were stored in a room at 28 with 70% relativehumidity for weeks to obtain moldy corn containing about 3 mg/kg ZEA. Byadding moldy corn to replace normal corn in the basal diet, theconcentration of zearalenone in all treatment diets was about 1 ppm(Table 14). Basal diets also contained very low concentration of ZEA(about 65 ppb). ZEA concentration of the five different diets wasdetermined using the Zearalenone Assay Kits (Beacon Company).

TABLE 14 Experimental design and composition of diets Group NC PC TS1TS2 TS3 Zearalenone(ppm) 0.065 1.05 1.12 1.08 1.10 Toxfin Supreme (g/kg)0 0 1 2 3 Ingredients, % Corn 53 53 53 53 53 Wheat middling 6.5 6.5 6.56.5 6.5 Soybean 24.76 24.76 24.76 24.76 24.76 Wheat flour 5 5 5 5 5 Fishmeal 5.5 5.5 5.5 5.5 5.5 Soybean oil 2.5 2.5 2.5 2.5 2.5 Lysine 0.3 0.30.3 0.3 0.3 Met 0.1 0.1 0.1 0.1 0.1 Thr 0.8 0.8 0.8 0.8 0.8 Dicalciumphosphate 0.3 0.3 0.3 0.3 0.3 Limestone 0.2 0.2 0.2 0.2 0.2 1% premix 11 1 1 1 Salt 1 1 1 1 1 Composition of nutrients DE, KCal/kg 3417 34173417 3417 3417 Crude protein, % 20 20 20 20 20 Crude fat, % 5.4 5.4 5.45.4 5.4 Ca, % 0.82 0.82 0.82 0.82 0.82 P, % 0.42 0.42 0.42 0.42 0.42*Premix (weight/kg): Mn 40 mg, Fe 130 mg, Zn 130 mg, Cu 15 mg, I 0.35mg, Se 0.3 mg, V_(A) 11,025 IU, V_(D3) 22.03 IU, V_(E) 80 IU, V_(K) 4.4mg, V_(B1) 4.4 mg, V_(B2) 11 mg, pantothenic 35 mg, Nicotinic acid 59.5mg, Chlorine 330 mg, Folic acid 0.9 mg, Biotin 0.5 mg and V_(B12) 55 μg.

Growth Performance.

Body weights and feed consumption were recorded at day 1 and 42 tocalculate average daily feed intake (ADFI), average daily weight gain(ADG) and feed conversion ratio (FCR).

Zearalenone Binding Efficacy Test.

ZEA was measured in feces, for which total feces of each group werecollected and weighed at 21, 22, 23, 40, 41 and 42 days. After thoroughhomogenization, about 10% of total feces were stored at −20° C. forfuture analysis. During the same period, the feed consumption of eachgroup was recorded to calculate the amount of ZEA consumed by pigletsaccurately. Toxfin Supreme binding efficacy on ZEA in vivo was expressedas a percentage, and calculated as the ratio of total zearalenonecontent in feces compared to total ZEA intake. Analysis of ZEA wasperformed according to the manual of the assay kit (Beacon Company).

Serum β-Estradiol.

At day 1, 21 and 42, three blood samples from each group were collectedvia the external pig tail vein. Analysis of serum β-estradiol wasperformed according to the manual of the assay kit (JianchengBio-technology Institute, Nanjing, China).

Vulva Size Measurement.

The vulva length and width of each piglet was measured using verniercaliper at day 1, 21 and 42. The vulva area was calculated according tothe formula: area=(lengthxwidth/2).

Histological Analysis.

At the end of the experiment, 3 piglets from each group were euthanized.The ovaries and uterus were removed immediately and then fixed inBouin's solution. They were then embedded in paraffin and sectioned at 5μm. Sections were finally stained with hematoxylin and eosin (H&E) andthe number of follicular and acinus cells were calculated. Thehistopathological examination was done under light microscopy by theexperimental pathology laboratories (Department of Veterinary Sciences;Southwest University, Chongqing, China).

Statistical Analysis.

Data were subjected to Levene's homogeneity of variances test before theanalysis for group differences. Data were processed by one-way analysisof variance (ANOVA) followed by Duncan's test using SPSS 16.0.Differences were considered to be significantly different at P<0.05.

Results

Growth Performance.

There were no differences in ADFI and FCR of piglets among all groups,and compared to the negative control group, the ADG of pigletssignificantly decreased in the PC group (Table 15). The addition ofToxfin Supreme in ZEA contaminated diets at 1, 2 and 3 g/kg all improvedADG of piglets and brought them back to same ADG as the negative controllevel (Table 15). Toxfin Supreme had no effect on ADFI and FCR, buthelped in the recovery of ADG of piglets fed ZEA contaminated diets atall three concentrations.

TABLE 15 Growth performance of piglets fed with different diets Group NCPC TS1 TS2 TS3 ADG  0.61 ± 0.02^(a)  0.56 ± 0.02^(b)  0.58 ± 0.03^(ab) 0.59 ± 0.01^(ab)  0.60 ± 0.03^(ab) (kg/day) ADFI 1.29 ± 0.09 1.24 ±0.06 1.29 ± 0.10 1.28 ± 0.05 1.34 ± 0.11 (kg/day) FCR 2.13 ± 0.12 2.22 ±0.08 2.21 ± 0.09 2.16 ± 0.09 2.24 ± 0.10 (g:g) Note: the values in thesame row with different superscript letters differ significantly (p <0.05)

In Vivo ZEA Binding Efficacy of Toxfin Supreme.

Fecal samples were collected and analyzed for presence of ZEA. Innatural ZEA contaminated feed, about 35% of ZEA could be eliminate infeces (Table 16). Toxfin Supreme significantly increased bindingcapability to ZEA in a dose dependent manner, being 64% in TS 1, 77% inTS2 and 92% TS3 (Table 3).

TABLE 16 In vivo zearalenone binding efficacy of Toxfin Supreme (%) Time(day) PC TS1 TS2 TS3 21-23 35.45 ± 2.66^(a) 65.04 ± 2.97^(b) 77.86 ±6.97^(c) 95.02 ± 1.87^(d) 40-42 36.04 ± 1.83^(a) 63.62 ± 1.22^(b) 76.18± 4.80^(c) 90.09 ± 3.65^(d) Aver- 35.74 ± 1.10^(a) 64.32 ± 1.52^(b)77.02 ± 1.21^(c) 92.55 ± 3.08^(d) age Note: the values in the same rowwith different superscript letters differ significantly(p < 0.05)

β-Estradiol Concentration.

At days 21 and 42, β-estradiol concentration in serum of the PC groupwas about 52% and 41% lower than the concentration found in the NC group(Table 17). These data showed that ZEA significantly decreased theestradiol secretion in weaned piglets. Compared to the PC, β-estradiolconcentrations in Toxfin Supreme treated piglets group TS 1 (1 g/kg)increased but were still lower than those in the NC group (Table 17).β-estradiol concentration was significantly increased and reached NClevels in the Toxfin Supreme treated groups TS2 (2 g/kg) and TS3 (3g/kg) (Table 4). Toxfin Supreme had a better efficacy as a ZEA binderwhen given at higher doses.

TABLE 17 Serum β-estradiol concentration of piglets at day 1, 21 and 42(ng/L) Time (day) NC PC TS1 TS2 TS3 1 21.59 ± 4.46  21.87 ± 5.44  20.96± 3.99  22.91 ± 2.49  21.69 ± 3.02  21 23.97 ± 3.92^(a) 12.46 ± 0.84^(c)14.13 ± 1.60^(c) 18.20 ± 1.94^(b) 19.78 ± 1.25^(b) 42 28.67 ± 5.71^(a)11.83 ± 1.37^(c) 17.90 ± 2.28^(b) 26.51 ± 1.04^(a) 23.81 ± 1.56^(a)Note: the values in the same row with different superscript lettersdiffer significantly (p < 0.05)

Vulva Size Measurements.

Vulva size of each piglet was monitored at day 1, 21 and 42. Vulva sizein this trial was expressed as a percentage and was calculated as theratio of vulva size at day 1 and 21, and day 1 and day 42 in each group.At both 21 and 42 days, the enlargements of vulva size in TS2 and TS3group were significantly less than those of PC group and TS 1 group. Thesize of vulvas in TS3 group was similar to those of NC group (Table 18).Toxfin Supreme could alleviate ZEA induced vulva size enlargement at adosage of 2, 3 g/kg (Table 18).

TABLE 18 Ratio of vulva area of piglets fed a ZEA contaminated dietbetween 1 and 21, 42 days of age (%) Time (day) NC PC TS1 TS2 TS3 21174.40 ± 42.23^(a) 412.55 ± 64.35^(c) 317.02 ± 62.06^(c) 252.07 ±43.85^(b)  220.41 ± 54.37^(ab) 42 198.07 ± 47.04^(a) 478.36 ± 62.04^(c)374.46 ± 47.93^(c) 296.75 ± 53.69^(bc) 240.40 ± 50.94^(ab) Note: thevalues in the same row with different superscript letters differsignificantly (p < 0.05)

Histological Analysis.

Tissue samples from varies and uterus were observed through lightmicroscopy and the relative amount of follicular and acinus cells foreach group were recorded (Table 19). These results indicated that thenumber of acinus increased significantly in TS2 and TS3 group comparedto those of PC group. Toxfin Supreme at a dosage of 2-3 g/kg alleviatedthe effects of ZEA on the development of sow's ovaries and uterus.

TABLE 19 Relative amount of follicular and acinus cells from ovaries anduterus from piglets' fed zearalenone contaminated diets (cells/cm²)Group NC PC TS1 TS2 TS3 Folliculus 5379 ± 1185^(a) 2832 ± 766^(b) 2657 ±564^(b) 3859 ± 890^(ab) 3843 ± 519^(ab) Acinus 1309 ± 146^(a)  542 ±37^(b)  873 ± 148^(b) 1265 ± 268^(a ) 1298 ± 247^(a ) Note: the valuesin the same row with different superscript letters differ significantly(p < 0.05)

Discussion

In this experiment, ˜1 ppm ZEA decreased ADG of piglets but had nosignificant effects on ADFI and FCR. The negative effects of mycotoxinadsorbents on depressing animal growth are always considered becauseadsorbent may also bind certain nutrients such as vitamins and mineralsas it has been shown when evaluated in vitro (Tomasevic-Canovic M,Dakovic A, Markovic V. 2000. Adsorpton effects of mineral adsorbents;Part III: Adsorption behaviour in the presence of vitamin B6 andmicroelements. Acta Veterinaria, 50(1):23-30). In this study, ToxfinSupreme did not show negative effects on performance and even exhibiteda slight improvement in ADG of piglets. It may be the concentration ofminerals and vitamins in commercial animal feeds is relative enough andthe dosage of absorbents added in feeds is very small.

Zearalenone is well known as a full agonist for estrogen receptors dueto the very similar chemical structure to estradiol. One ppm ZEA causedobvious visual hyperestrogenism effects on female weaned piglets such asswelling of the vulva after 3-6 weeks exposure. Exposure to ZEA treateddiets for 3 and 6 weeks, the excretion of estradiol was depressedsignificantly compared to those levels excreted through control piglets.The estradiol concentration in serum was recovered to normal levels whenToxfin Supreme was added into zearalenone contaminated diets at 2, 3g/kg feed. As shown in this experiment, about 75-90% zearalenone intakeby piglets was adsorbed by Toxfin Supreme (2-3 g/kg) and then eliminatedthrough feces. This is why Toxfin Supreme may help improve estradiolexcretion and may alleviate ZEA-induced clinical signs such as red andswelling of piglet vulva.

Example 8 Material and Methods

Experimental Animals.

Twenty-four-old day and mixed sex free-range broilers of a commercialstrain (T44×SA51) were obtained from a commercial hatchery. They wereindividually weighed and randomly distributed to different wire-flooredcages. The average initial weight was 417.35±45.26 g (mean of 200 birds)with a room temperature of 20±5° C., natural illumination and manualventilation.

Feed formulation. Animals were fed a commercial, unmedicated,corn-soybean meal basal diet that contained or exceeded the levels ofcritical nutrients recommended by the National Research Council (1994).Feed ingredients used in formulating the control diet did not containmycotoxins at detectable levels. The basal control diet was formulatedas shown in Table 20.

TABLE 20 Feed ingredients used in formulating the basal diet. Percentagecomposition of the basal diet (g/100 g). Ingredient Percentage of diet(g/100 g) Wheat 50.00 Soybean meal 25.64 Maize 12.00 Barley 5.00 Soybeanoil 1.00 Dicalcium phosphate 1.00 Fish meal 1.00 Calcium carbonate 0.90Animal fat 2.00 Salt 0.25 Sodium bicarbonate 0.13 *Vitamin/mineralpremix 0.40 Methionine 0.30 Lysine 0.30 Threonine 0.08 Total 100.00Approximate analysis Dry matter 89.63 Total protein 19.30 Ash 5.20 Fat5.00 Fiber 3.00 Lysine 1.13 Methionine 0.28 Yeast and mold count (cfu/g)3.6 × 10³ *Vit. A 12500 UI; Vit. D 33500 UI; tocopherols 4000 UI; Vit.K3 800 mg; folacin 200 mg; thiamine 600 mg; riboflavin 1800 mg; niacin6000 mg; calcium panthothenate 2000 mg; pyridoxine 600 mg;cyanocobalimin 4 mg; biotin 8 mg; manganese 30,000 mg; zinc 20,000 mg;iodine 480 mg; cobalt 80 mg; selenium 40 mg; BHT 25,000 mg; anti-cakingagent 6000 mg.

Diet Preparation.

The binders are commercial products sold by Kemin Europe N.V. under thetrademarks Toxfin (coded as B1) and Toxfin Supreme (coded as B2); Toxfinis a blend of sepiolite and bentonite and Toxfin Supreme is a blend ofleonardite and sepiolite. Four mycotoxigenic fungi cultures, containingAflatoxin B1 (coded as AFB1), Fumonisin B1 (coded as FB1), Ochratoxin A(coded as OA) and T-2, were obtained from Kemin Europe N.V. The toxinswere incorporated into the basal diet by mixing the appropriatequantities with 2 Kg diet and then mixed with the rest of the basal dietin a paddle feed mixer to produce the desired concentrations (Table 21).To test the efficacy of binders as dietary treatments for mycotoxicoses,the mycotoxin contaminated diet was supplemented with 3000 ppm of B1 andanother diet supplemented with 3000 ppm of B2 (Table 21). Binders werethoroughly shaken for 30 seconds by hand prior to dosing the feed andmixed the appropriate quantities with 2 Kg mycotoxin-contaminated dietand then mixed with the rest of contaminated diet in a paddle feed mixerto produce the desired treatments.

Different samples of the mycotoxin contaminated feed were analyzedbefore starting the trial to check final OA concentration by HPLC as anindicator of homogeneity.

Experimental Design.

Animals were randomly allocated to different treatment groups and housedin 4 cages (10 chickens per cage). Treatments were replicated fivetimes. Individual boxes into cages contained a water trough and afeeder. The birds were allowed to acclimatize to the cages environmentfor 1 day before the commencement of the feeding trial. The animals weresupplied feed and water ad libitum. Feed consumption was recorded dailyfor each cage. Animals were weighed at the end the week (28-day-oldbirds) and then they were euthanized by CO₂.

TABLE 21 Mycotoxins and binders levels contained in differentexperimental diets FB₁ AFB1 OA T-2 B1 B2 Amount 989.47 29.79 16.79 11.85287.37 287.37 Toxin 1900 800 1400 1982.61 Concentration Amount 329.839.79 5.60 3.95 95.80 95.80 added to batches (g) Final concentration infeed T1 ¹BLD BLD BLD BLD BLD BLD T2 20 ppm 250 ppb 250 ppb 250 ppb   0  0 T3 20 ppm 250 ppb 250 ppb 250 ppb 3000   0 ppm T4 20 ppm 250 ppb 250ppb 250 ppb 3000 ppm ¹BLD: below detectable limit

Collection of Feces Samples.

Broilers were given ad libitum access to the control diet during 1-dadaptation to the cages followed by a 3-d test in each replicate. Each5-d test period consisted of 1-d of adjustment, 3-d of experimental dietfeeding, and 1-d for change-over; test diets were fed continuouslyduring 3-d test period. After the morning feeding on the change-overday, fecal samples (approximately 400 g of wet feces per treatment) werecollected from the stainless steel floor from behind animals, stored inplastic packs under refrigeration conditions and shipped to a certifiedlaboratory for mycotoxin assay. The wet samples were immediatelylyophilized after they were received by the laboratory.

Mycotoxin Quantification in Feces.

Samples of Broiler Feces.

Excreta were collected as described in the experimental design sectionand sent to the laboratory in the same day. They were weighed(389.9±69.1 g) and lyophilized. Samples were re-weighed for moistureestimation and crushed-mixed with mortar and pestle. All samples werefrozen and stored at −20° C. until required.

Apparatus and Reagents.

HPLC grade acetonitrile and methanol were purchased from Lab-Scan(Dublin, Ireland) and HPLC grade acetic acid from Merck (Darmstadt,Germany). Ultrapure water was obtained from a Milli-Q Plus apparatusfrom Millipore (Milford, Mass.). The immunoaffinity columns RIDAochratoxin A were supplied by R-Biopharm AG (Glasgow, Scotland).Aflatoxin B1; fumonisin B1 and ochratoxin A standards were provided bySigma (St. Louis, Mo.) and stock solutions (0.01 mg/ml) were prepared inmethanol and stored at −21° C. Reagents for sodium bicarbonate buffer pH8.1 (0.13 M) and PBS phosphate buffer saline pH 7.4 (20 mM) wereprovided by Panreac (Barcelona, Spain).

The LC system consisted of an Agilent Technologies 1100 high-performanceliquid chromatogram coupled to an Agilent fluorescence detector at: (1)365 nm (excitation) and 455 nm (emission) for aflatoxin B1; (2) 335 nm(excitation) and 440 nm (emission) for fumonisin B1; (3) 333 nm(excitation) and 460 nm (emission) for ochratoxin A. The LC column wasAce 5 C18, 250×4.6 mm, 5 μm particle size (Advanced ChromatographyTechnologies, Aberdeen, Scotland). The isocratic LC mobile phases were:(1) distilled water, acetonitrile and methanol (55:30:15 v/v/v); theflow rate: 1.2 ml/min for aflatoxin B1; (2) distilled water,acetonitrile and acetic acid (51:48:1 v/v/v); the flow rate: 1.2 ml/minfor ochratoxin A; (3) distilled water, acetonitrile and acetic acid(53:46:1 v/v/v); the flow rate: 1.2 ml/min for fumonisin B1.

Analysis of Aflatoxin-B1, Ochratoxin-A and Fumonisin-B1 in BroilerFeces.

The analytical method used in this trial has been previously describedby Ariño et al., (2007) with some modifications. Briefly, (1)Aflatoxins-B1: 5 g of broiler feces was mixed with 25 ml methanol 70%;(2) Ochratoxin-A: 5 g of broiler feces was mixed with 20 ml methanol70%; (3) Fumonisin-B1: 10 g of broiler feces was mixed with 20 mlmethanol 75% using an homogenizer for 15 min. The extract wascentrifuged at 3500 rpm during 15 min and filtrated by Whatman n° 1. Theaflatoxin and ochratoxin filtrates were purified through the RIDA toxinimmunoaffinity. Immunoaffinity cleanup was done according to theinstructions of the manufacturer: equilibrate with 2 ml PBS/methanol(90/10), pass sample extract slowly and continuously through the column,rinse the column with 10 ml PBS/methanol (90/10), and eluate with 2 mlmethanol. The different toxins eluates were collected into an opaquevial. The eluates were evaporated to dryness in a heating block under agentle stream of nitrogen, and re-dissolved in the methanol:distilledwater:acetic acid, (30:69:1 v/v/v)

The fumonisin filtrate was eluted in a SPE vacuum manifold (SAX 500 mg).The cartridges were previously conditioned with 5 ml of methanol 75%followed by 5 ml of distilled water at a flow rate of 1 ml/min, beforeloading 10 ml of the sample solution at a flow rate of 0.8 ml/min.Subsequently cartridges were washed methanol 75% followed by methanol100%. Fumonisin hold in the cartridges were eluted in amber tubescontaining acetic acid:methanol (1:99 v/v) and the eluate was evaporatedto dryness in a heating block under a gentle stream of nitrogen, andre-dissolved in the injection solvents (acetonitrile:distilledwater:acetic acid, 46:53:1, v/v/v).

Aflatoxins and fumonisins aliquots of 20 μl and ochratoxin aliquots of100 μl were injected into the LC-FLD system. Identity of ochratoxin waschemically confirmed by methyl ester formation according to Li et al.(1998). Identity of aflatoxin and fumonisin was chemically confirmedaccording UNE-EN 14123 and UNE-EN 13585. The analytical method wasvalidated in-house with respect to precision and recovery. The averagerecovery and relative standard deviation (RSDr, repeatability) obtainedby the described method were 84% for OA; 75% for AFB1 and 90.6% for FB1,(RSD 5%, 5%, 3.8%, respectively). The performance characteristics werewithin the acceptable margins indicated in the Commission Regulation No.401/2006 (EC, 2006). The study of sensitivity for aflatoxin, ochratoxinand fumonisin indicated that the limits of quantification (LOQ) were:0.2 ng/g; 2 ng/g and 200 ng/g respectively.

Analysis T-2 Toxin in Broiler Feces.

The T-2 toxin was analyzed by ELISA technique (Ridascreen®). An amountof 5 g of broiler feces was mixed with 25 ml methanol 70% andhomogenized (magnetic stirrer) for 10 min. The extract was centrifugedat 3500 rpm during 15 min and filtrated by Whatman n° 1. Total volumesof 50 μl of the extract were diluted with 950 μl of PBS buffer. Samplesand the standards were used according to the manufacturers'instructions. The measurement was made photometrically at 450 nm anddetection limit was <5 ppb (approx. 3.5 μg/kg).

Statistical Analyses.

All data were analyzed by ANOVA using the SPSS procedure (SPSS version15.0; 2006). Treatment means with significant differences were ranked byTukey's multiple range test. Pearson's correlation coefficients werecalculated among variables. All statements of differences were based onsignificance at p<0.05.

Results

Clinical Effects.

At day 1, the excreta from all birds had a normal consistency. Thiscondition began to change until day 3 except for control batch, whenexcreta began to be sticky and adhered to the wire mesh pen floors andaround the area of the birds. Previous reports have associated diarrheawith fumonisin toxicity after 4-13 days infected fed. Sticky excretafrom broilers fed Fusarium-infected corn has been reported; a depressionin diet dry matter digestibility and diarrhea was associated with thisphenomena. The data in the present study indicate qualitativedescription in the faeces consistency, however fecal moisture content atday 3 was not significantly different (p>0.05) among treatments (Table22). These values are considered normal. No mortality was observed dueto diets.

TABLE 22 Fecal moisture content in broilers fed different diets for 3days Moisture (%) T1 (control) 63.0 ± 11.0 T2 (mycotoxins) 64.0 ± 8.6 T3 (mycotoxins + B1) 61.6 ± 10.7 T4 (mycotoxins + B2) 65.0 ± 7.3  Valuesrepresent the x SD of five groups of ten broilers per treatment Eachvalue is a mean of five determinations

Consumption and weight gain. Initial and final weights of birds fedexperimental diets are presented in Table 23. There was no statisticaldifference in body weight. However, final weight of birds and feedintake fed the T2 diet (mycotoxin contaminated feed) were respectively2% and 1% less than those of control batch (p>0.05). Conversely, birdsfed binders showed higher final body weight and feed consumption thanthose birds of control batch (p>0.05).

TABLE 23 Influence of experimental diets on body weight and total feedintake of broilers fed for 3 days Total feed Feed intake Body Weight(g/bird) intake ¹Change difference 24 day old 28 day old (g/10 birds; 3d) (%) from control (%) T1 415.9 ± 53.84 448.5 ± 65.5 1266.4 ± 139.8 0 0T2 416.7 ± 45.75 440.3 ± 58.7 1253.2 ± 119.8 −2 −1 T3 418.0 ± 43.23463.7 ± 53.9 1318.0 ± 121.3 +3.4 +4.1 T4 418.7 ± 39.25 461.9 ± 68.51334.8 ± 177.7 +3.0 +5.4 Sig (*) NS NS NS (*) NS: no significantdifferences (p > 0.05) Values represent the x ± SD of five groups of tenbroilers per treatment ¹Change = percentage change in body weightrelative to controls

Mycotoxins in Excreta.

Table 24 shows different toxin concentrations in the excreta of birdsafter 3 days of feeding experimental diets. Results showed aflatoxin B1concentration in excreta of birds fed B1 was 66% significantly higher(p<0.05) than birds fed T2 diet. Aflatoxin B1 concentration in excretaof birds fed B2 was 26% significantly higher (p<0.05) than those birdsfed T2 diet. Fumonisin B1 concentration in excreta of bird fed B1 and B2were respectively 52% and 8% higher to birds fed T2 diet. Although theprecise mode of action of binders (B1 and B2) is not known, wehypothesized that binders might partially trap the aflatoxin andfumonisin molecules in its matrix preventing a partial aflatoxin andfumonisin absorption from the gastrointestinal tract. So, B1 and B2administered concomitantly with a pool of mycotoxins were shown toadsorb AFB1 and FB1, thereby limiting AFB1 and FB1 bioavailability andincreasing excretion in birds. However, binders had no effect onochratoxin A and T-2 toxin excretion after 3 days of diet exposure. T-2toxin and OA toxic effects have not been found to be reduced by anysorbent up to now (Kubena et al., 1990; Huff, W. E., L. F. Kubena, R. B.Harvey, and T. D. Phillips. Efficacy of hydrated sodium calciumaluminosilicate to reduce the individual and combined toxicity ofaflatoxin and ochratoxin A. Poult. Sci. 71: 64-69. 1992; Bailey, R. H.,L. F. Kubena, R. B. Harvey, S. A. (1998). Buckley, and G. E.Rottinghaus. Efficacy of various; sorbents to reduce the toxicity ofaflatoxin and T-2 toxin; in broiler chickens. Poult. Sci. 77:1623-1630;García, A. R.; Avila, E.; Rosiles, R.; Petrone, V. M. (2003). Evaluationof two mycotoxin binders to reduce toxicity of broiler diets containingOchratoxin A and T-2 toxin Contaminated Grain. Avian Diseases47:691-699).

New binding products have appeared in an attempt to counteractmycotoxins (Hoerr, F. J., W. W. Carlton, J. Tuite, R. F. Vesonder, W. K.Rohwedder, and G. Szigeti. Experimental thricothecene mycotoxicosisproduced in broiler chickens by Fusarium sporotrichiella var.sporotrichioides. Avian Pathol. 11:385-405. 1982; Devegowda, G., and M.V. L. N. Raju. Influence of esterified-glucomannan on performance andorgan morphology, serum biochemistry and haematology in broilers exposedto individual and combined mycotoxicosis (aflatoxin, ochratoxin and T-2toxin). Br. Poult. Sci. 41:640-650. 2000); however, they have not beenproperly evaluated since most of them have only received in vitroevaluation, which does not provide reliable information about thesorbent activity under field conditions. This has to be considered notonly in a sorbent evaluation process, but also in the source ofmycotoxin, because many evaluations have consisted of pure mycotoxinadded to feed. Under realistic conditions, the contaminated grainincludes many other metabolites, most of them unidentified, whichdefinitively have effects on birds. Different research have emphasizedthe differences between the toxic effects observed in birds fed puremycotoxin contaminated diets and those fed diets contaminated withmycotoxigenic fungi cultures (Hoerr et al., 1982; Rotter, R. G., A. A.Frohlich, R. R. Marquardt, and D. Abramson. Comparison of the effects oftoxin-free and toxin containing mold contaminated barley on chickperformance. Can. J. Anim. Sci. 69:247-259. 1989). At present, however,the utilization of mycotoxin-binding adsorbents is the most applied wayof protecting animals against the harmful effects of decontaminatedfeed. So far, no single adsorbent was tested to be effective againstmost types of mycotoxins. However, the addition of different adsorbentsto animal feed provides versatile tools of preventing mycotoxicosis.

TABLE 24 Mycotoxin concentration in feces detected by HPLC¹ after 3 daysof diet exposure. (results expressed as ng/g of lyophilized feces) T1 T2T3 T4 Sig (*) Aflatoxin B₁ (ppb)  BDL² 18.8^(a) ± 2.6  23.7^(b) ± 1.920.4^(ab) ± 2.8  * Ochratoxin A (ppb) BDL  55.0 ± 17.7  51.7 ± 4.0  60.0± 10.4 NS Fumonisin B₁ (ppb) BDL 545.8^(a) ± 207.1 910.0^(b) ± 98.4823.5^(ab) ± 119.0 * T-2 (ppb) BDL 115.6 ± 4.4    123.8 ± 19.0  105.2 ±18.8 NS Each value is a mean of five determinations + SD File valueswith different superscripts are significantly different ¹HPLC was usedto determine mycotoxins, except for T-2 which was confirmed by ELISA²BLD means below detectable limit

Conclusions

The results obtained in our study indicated that feed consumption wasnot affected by dietary inclusion of mycotoxin contaminated diets(p>0.05) after 3 days of exposure. Supplementation with binders did nothave any effect on feed consumption and weight gain.

B1 and B2 administered concomitantly with pool of mycotoxins were shownto adsorb effectively AFB1 and FB1, thereby limiting AFB1 and FB1bioavailability and increasing excretion in birds.

Example 9 Material and Methods

Animals and Feeding.

In experimental design 60 gilts (Topigs) were housed in individual pensadjacent to each other in the same shed in groups of 15 animals. Giltswere allowed ad libitum access to food and water. Ingredientscomposition is shown in Table 25.

TABLE 25 Composition of diets used to determine the effect of adsorbentson zearalenone toxicosis in prepubertal gilts Control ZEA ZEA + NT ZEA +ET Pure Zearalenone (ppm) 0.02 1.03 1.14 1.05 Vomitoxine (ppm) 0.30 0.310.36 0.27 NT (ppm) 0 0 3000 0 ET (ppm) 0 0 0 3000 Ingredient Percentageof diet^(a) Barley 35.00 36.00 35.50 34.00 Wheat 32.53 33.00 31.50 34.00Soybean (FF Danex) 7.97 7.97 7.97 34.20 Soybean (47/5 Brasil) 11.0011.05 12.00 11.30 Sugar beet pulp 8/7.5 2.00 3.00 2.00 2.50 Fat 2.302.26 2.34 2.36 Treonine 0.00 0.00 0.00 0.00 Tryptophan 0.02 0.02 0.030.02 Lysine 50% Liquid 0.05 0.05 0.05 0.04 Dicalcium phosphate sa 0.200.20 0.20 0.20 Vitazym 02 0.10 0.10 0.10 0.10 Salbiotic 0.30 0.30 0.300.30 Visp vitaprotein 50pl 2.50 2.50 2.50 2.50 Vitapops 6.00 6.00 6.006.00 Proximate analysis (%)^(b) Moisture 9.76 9.98 9.83 9.93 Protein18.73 18.18 18.59 18.32 Fiber 4.33 4.45 4.43 4.20 Fat 6.81 6.51 6.656.72 Ash 5.45 5.33 5.56 5.54 Strach 39.08 40.21 39.79 39.66^(a)Percentages expressed on dry matter. ^(b)The subset percentages donot always add up to 100% because not all components were determined

The pure toxin and the commercially available adsorbents, sold under thetrademark Toxfin (coded as NT) and Toxfin Supreme (coded as ET), weresupplied by Kemin Europe N.V.; Toxfin is a blend of bentonite andsepiolite and Toxfin Supreme is a blend of sepiolite and leonardite, amineraloid high in humic substances. They were included into theexperimentally contaminated diet which resulted in a dietary ZEAconcentration of approximately 1 ppm. The control diet contained 0.02ppm of ZEA (from raw materials) and any adsorbent (Table 25). The ZEA+NTand ZEA+ET diets contained approximately 1 ppm of ZEA and 3000 ppm of NTand ET, respectively. Different diets were fed to piglets during 6weeks. Total feed intake was recorded for the duration of trial. Fromthe performance data, daily gain (kg/day), daily feed intake (kg/day),and feed efficiency (gain/fed intake) describing the entire test periodwere calculated.

Determination of Zearalenone in Feed.

An AOAC-approved method for the determination of zearalenone in feedsamples by HPLC and TLC was used (Scott, P. M., T. Panalalrs, S. Kanhcreand W. F. Miles. 1978. Determination of zearalenone in cornflakes andother com-based foods by thin layer chromatography, high matography/highresolution mass spectrometry. I.AOAC 61593). For calculating the valuesof zearalenone, duplicate determinations were evaluated from 10different mixings of feed.

Clinical Evaluation.

All prepubertal gilts were observed weekly for signs ofhyperestrogenism. Vulvas were assessed for swelling, redness andenlargement characteristics of estrogenic stimulation using a subjective5 point scale (1=least stimulated; 5=most stimulated scale).

All gilts tested were apparently healthy with no signs of clinicalinfection. Respiratory and heart rate were normal. Mammary glands,vulva, mucosal, feces and urine aspect were normal.

Using serology test, the piglets were classified as negative for allmajor piglet pathogens except for Circovirus. Results were not availablein this study but certified by the farm of the origin.

Blood Samples.

Blood was collected via the external pigtail vein into a tube containingEDTA anti-coagulant for hematology and another tube without EDTA forbiochemical analysis. Blood collection was performed at the 0, 3^(rd)and 6^(th) week and coincided with the weighing of animals. Analyseswere performed immediately after blood collection. A fraction of theblood collected in dried tube was centrifuged at 2000 g for 15 min atroom temperature. The serum yield was transferred into Eppendorf tubesand used for some biological parameters. Blood cell count and routinehematological analysis were performed using an automated cell counter,with adapted dilution. Determinations enzymatic activity were achievedin duplicates and results expressed in International Units/Liter (IU/L).

Estradiol-17-β (E2).

Serum estradiol-17-β (E2) concentrations were determined by asolid-phase, competitive chemiluminescent enzyme immunoassay(Immullite®). The solid phase (bead) is coated with rabbitanti-estradiol plyclonal antibody validated previously. The reagentcontains alkaline phosphatase (bovine calf intestine) conjugated toestradiol. The estradiol enzyme conjugate competes with estradiol in thesample for limited antibody-binding sites on the bead. The excess sampleand reagent are removed by a centrifugal wash. Finally chemiluminiscentsubstrate is added to the bead and signal is generated in proportion tothe bound enzyme. Results were expressed in pg/ml and the analyticalsensitivity was 15 pg/ml.

Urine and Feces Samples.

Six animal of each pen were placed in individual metabolism cages in aroom at 27±2° C. for 20 consecutive days; 1 week of adaptation to cagesand 4 weeks (4 days per week) for daily records of consumption and fecaland urinary collection. At the end of about 12 hours the animals wereremoved in turns of three from the cages and returned to the pens.Samples were taken daily. The feces and urine collection was about 10%of total excretion. Samples were frozen and sent by transport to alaboratory to determine the presence of metabolites derived from ZEA.

Slaughter.

After 3 weeks of diet-exposure, 5 piglets of each treatment pen wereslaughtered on the same day, in the same slaughterhouse, located lessthan 50 km from the farm, in commercial terms, according to Europeanstandards. Animals were stunned with CO and subsequently exsanguinated.Uterus, kidneys and liver were excised and weighed. Approximately 25 gof fresh liver and kidney were place into a formalin flask. Immediatelyafter sacrifice, the reproductive organ was retrieved, identified andplaced individually in phosphate-buffered saline (PBS, Sigma-Aldrich,Madrid) to avoid drying of the structures for subsequent evaluation.About 5 ml of bile of each slaughtered animal were collected into a tubeto analyze ZEA concentration for better ZEA degradation understanding.Bile tubes were sent by transport to the laboratory in refrigerationconditions. All samples were stored and transported in coolers todifferent laboratories. After 6 weeks, all the remaining animals wereslaughtered and samples were taken in the same conditions describedabove. Carcasses were seized and properly destroyed.

Histopathology Examination.

Each female was examined for gross anomalies on selected organs (liver,kidneys, ovaries and uterus). The organs were weighed and measured. Themacroscopic, microscopic, and morphometric aspects were valued. Selectedtissues and all gross lesions were fixed in 10% neutral bufferedformalin, embedded in paraffin, sectioned at 3-5 μm, stained withhematoxylin and eosin, and examined by light microscopy. Thehistopathological examination of the fixed tissues was done by auniversity pathology laboratory.

Data Analysis.

Two-way analysis of variance was performed on blood parameters, ovary,oviduct, cervix, uterine horns weight and vagina, ovary, oviduct,cervix, uterine horns size. The factors or independent variablesconsidered in this analysis were treatment (four levels: no ZEA added, 1ppm ZEA, 1 ppm ZEA+3000 ppm NT and 1 ppm ZEA+3000 ppm ET) and time (twolevels: 3 and 6 weeks of exposure). The treatment×time interactions wereused to determinate whether time has any significant effect onZeralenone/adsorbents toxicosis effect. Differences among treatments ortime exposure were analyzed by Student's t-test. Chi-square test wasused in order to measure the association among categorical variables ofthe study. All of the statistical analyses were performed with SPSS(Statistical Package for the Social Science for Windows, version 15.0,2006).

Results

Weight Gain, Feed Consumption and Feed Efficiency of Animals.

TABLE 26 Mean and standard deviation of gilt weight evolution. Weight(kg) Time (weeks) Control ZEA ZEA + NT ZEA + ET Sig (*) 0 16.96 ± 0.98 16.36 ± 1.4  16.18 ± 1.98 15.53 ± 1.92 NS 3 27.7 ± 2.12 26.07 ± 2.2826.61 ± 2.04 25.38 ± 1.83 NS 6 34.4 ± 2.29  36.2 ± 2.69 36.38 ± 1.79 34.4 ± 3.68 NS (*) NS = Not significant

TABLE 27 Summary of productivity traits in different batches. ControlZEA ZEA + NT ZEA + ET Sig ⁽*⁾ Daily feed intake 1.08 ± 0.09  1.20 ±0.02  1.01 ± 0.04  1.05 ± 0.11  NS (kg/day) Food efficiency 2.16 ± 0.3832.09 ± 0.194 1.74 ± 0.256 1.90 ± 0.651 NS (gain/feed intake; kg/kg)Daily gain 0.593 ± 0.11  0.589 ± 0.12  0.595 ± 0.14  0.598 ± 0.09  NS(kg/day) Oral ZEA intake  0.02 ± 0.001a 1.23 ± 0.01b 1.15 ± 0.03b 1.10 ±0.01b *** (mg/day per animal) ⁽*⁾ NS = Not significant (p > 0.05); ***(p < 0.001)

Neither Zearalenone nor adsorbents had any effect on weight gain ofgilts (Table 26). There were no differences neither feed consumption norfeed efficiency (Table 27). There were no differences among ZEA-treatedgilts for oral ZEA intake. These data support the findings of James andSmith (James, L. J.; Smith, T. K. (1982). Effect of dietary alfalfa onZearalenone toxicity and metabolism in rats and swine. Journal of AnimalScience, 55:110-118) who noted that in swine, ZEA had no significanteffect on growth characteristics.

Clinical Effects.

Within 3 weeks after diet-exposure to experimental diets, vulva scoresincreased. ZEA contaminated diets produced vulva scores greater thanthose of controls (FIG. 1). Enlargement increased in the followingsignificantly order: ZEA≧ZEA+NT>ZEA+ET ≧CON. Within 6 weeks ofdiet-exposure, effects were greater intensity to those observed at week3 (Table 28). Results demonstrated that ET and NT did not alleviate allZEA clinical effects but ET adsorbent was found to be more effective.

TABLE 28 Mean and standard deviation of vulva size of gilts after 6weeks of different diet-exposure. 6 weeks Control ZEA ZEA + NT ZEA + ETSig ⁽*⁾ Vulva size (cm) 1.45 ± 0.28^(a) 3.12 ± 0.52^(c) 2.81 ± 0.41^(bc)2.48 ± 0.30^(b) *** ⁽*⁾ ^(a-c)Within a row, value with a superscriptletter was significantly different (p ≦ 0.001)

Blood Analysis Results.

The metabolism of ZEA seems to occur essentially in the liver leading toα and β zearalenol, the latter being nontoxic.

TABLE 29 Mean and standard deviation of blood parameters on day 0. Time(0 days) Control ZEA ZEA + NT ZEA + ET Sig ⁽*⁾ Leucocytes 24500 ± 207219920 ± 2072 19249 ± 2072 20939 ± 2072 NS (count/mm³) ALP (IU/liter^(†))1002 ± 83  845 ± 83 895 ± 83 956 ± 85 NS GGT (IU/liter) 57 ± 8 59 ± 8 74± 8 73 ± 8 NS ALT ((IU/liter) 70 ± 6 65 ± 6 64 ± 6 56 ± 6 NS AST(IU/liter) 119 ± 62 181 ± 62 159 ± 62 213 ± 64 NS Estradiol-17-β (pg/ml)23 ± 2 26 ± 2 21 ± 2 21 ± 2 NS Time*Treatment NS ⁽*⁾ NS = Notsignificant. ^(†)ALP: alkaline phosphatase; GGT: γ-glutamyltransferase;ALT: alanine aminotransferase; AST: aspartate aminotransferase

In order to get a general view on the toxicology of zearalenone, mainlyliver, kidney and reproductive organ toxicity, other possible adverseeffects on blood enzymes or biological markers have been investigatedafter 3 and 6 weeks of exposure.

No differences were observed for blood parameters among animals at thebeginning of the studio, which indicated the same healthy status fordifferent batches (Table 29). However, ALP and AST activity of allbatches were over reference value (ALP≦300 IU/L; AST≦80 IU/L). GGTactivity was not significantly different among treatments butadsorbent-treated gilts showed the higher values (p<0.05).

Table 30 shows the influence of treatments on the haematologicalparameters in prepubertal gilts after 3 weeks of ZEA and adsorbents dietexposure. In the ZEA and ZEA+NT-treated gilts, the ALP activity wassignificantly lower than in control and ZEA+ET batches. As also shown inTable 30, adsorbent-treated prepubertal swine had higher GGT activitiesthan their non treated counterparts. No differences were found in anyother blood parameters among batches.

TABLE 30 Mean and standard deviation of blood parameters after 3 weeksof different diet-exposure. Time (3 weeks) Control ZEA ZEA + NT ZEA + ETSig ⁽*⁾ Leucocytes (count/mm³)  9854 ± 1346 13320 ± 1300 11719 ± 134612695 ± 1300 NS ALP (IU/l^(†))  954 ± 47^(c)  790 ± 45^(ab)  760 ±51^(a)  929 ± 64^(bc) * GGT (IU/l)   78 ± 10^(ab)  56 ± 9^(a)   96 ±11^(b)  107 ± 13^(b) * ALT (IU/l) 63 ± 4 55 ± 4 53 ± 4 50 ± 5 NS AST(IU/l) 100 ± 8  79 ± 8 90 ± 9  91 ± 11 NS Estradiol-17-β (pg/ml) 29 ± 225 ± 2 23 ± 3 30 ± 3 NS Time*Treatment NS ⁽*⁾ ^(a-b)Within a row, valuewith a superscript letter was significantly different (p ≦ 0.01); NS =Not significant. ^(†)ALP: alkaline phosphatase; GGT:γ-glutamyltransferase; ALT: alanine aminotransferase; AST: aspartateaminotransferase

ALP activity was significantly higher (p<0.05) in control batch after 6weeks of exposure (Table 31). GGT activity was not significantlydifferent among batches but adsorbents-treated prepubertal swine showedthe higher values again. The ALP and GGT differences found from 3 toweek 6, may suggest that there is a biological variation to recover fromstress among and within groups.

TABLE 31 Mean and standard deviation of blood parameters after 6 weeksof different diet-exposure. Time (6 weeks) Control ZEA ZEA + NT ® ZEA +ET ® Sig ⁽*⁾ Leucocytes (count/mm³) 3921 ± 665 3839 ± 665 4829 ± 7433920 ± 665 NS ALP (IU/l^(†)) 1093 ± 66^(b )  688 ± 66^(a)  683 ± 69^(a) 830 ± 66^(a) * GGT (IU/l)  64 ± 11  46 ± 11  56 ± 11  66 ± 11 NS ALT(IU/l)  63± 52 ± 6 63 ± 7 46 ± 6 NS AST (IU/l) 63 ± 7 63 ± 7 77 ± 8 67 ±7 NS Estradiol-17-β (pg/ml)  25 ± 3^(b)  20 ± 3^(a)  17 ± 3^(a)  26 ±3^(b) * Time*Treatment NS ⁽*⁾ ^(a-b)Within a row, value with asuperscript letter was significantly different **(p < 0.01); ***(p <0.001); NS = Not significant ^(†)ALP: alkaline phosphatase; GGT:γ-glutamyltransferase; ALT: alanine aminotransferase; AST: aspartateaminotransferase

Concerning ZEA, dietary concentrations of 1 ppm for 6 weeks of exposuredid not impair liver function and disturb blood parameters in ourexperiments.

Table 31 shows estradiol-17-β plasma (E2) concentration differences(p<0.05) at week 6. From high to E2 low plasma concentration, the orderbatch was: CON=ZEA+ET>ZEA=ZEA+NT. These results could suggest that ETbinds effectively to zearalenone and then mean plasma estradiolconcentration were similar to control group, indicating basal levels ofendogenous estradiol production for prepubertal gilts was maintained byET-supplementation after 6 weeks of exposure. It was found similarevolution for E2 plasma concentration in all batches (FIG. 2). There wassurge of E2 at week 3 and returned to basal levels at week 6. The slightE2 plasma concentration increase found in CON and ZEA+ET was notdifferent (p>0.05) to basal levels whereas it was different to ZEA andZEA+NT due to the significant E2 decrease at week 6. The resultsindicated that the hormonal profiles during the pubertal phase of giltswere modified by zearalenone in diet. In fact, maturation ofhypothalamo-hypophysial responsiveness to estradiol, culminating in theability to produce a preovulatory LH surge, occurs during theprepubertal period. The hypothalamo-hypophysial unit is sensitive toestrogens long before puberty occurs. For example, as early as 40 daysof age, LH increased in serum after estradiol benzoate (EB), but thepattern was one of multiple small surges that were less synchronous andlower in magnitude than in 160-days-old gilts (Fleming, M. W. and R. A.Dailey. 1985. Longitudinal study of the surge of gonadotropins inducedby exogenous hormones in prepuberal gilts. Endocrinology 116:1893). Dialet al. (Dial, G. D., O. K. Dial, R. S. Wkinson and P. J. Dziuk. 1984.Endocrine and ovulatory response of the gilt to exogenous gonadotropinsand estradiol during sexual maturation. Biol. Reprod. 30:289) reportedthat by 175 days of age, a single surge in LH similar to that of amature sow occurred in response to EB, but at younger ages results weresimilar to those of Fleming and Dailey (1985). Because zearalenoneaffects some estrogen target tissues, exposure to zearalenone prior topuberty potentially could alter estrogen-sensitive mechanisms involvedin maturation of the hypothalamus and the basal E2 concentration inplasma.

TABLE 32 Time effect of diet exposure on blood parameters for differentbatches. Control ZEA ZEA + NT ZEA + ET Leucocytes (count/mm³) *** ****** *** ALP (IU/l^(†)) NS NS NS NS GGT (IU/l) NS NS * * ALT (IU/l) NS NSNS NS AST (IU/l) ** ** * * Estradiol-17-β (pg/ml) NS * * NS * (p <0.05); ** (p < 0.01); *** (p < 0.001); NS = Not significant (p > 0.05)^(†)ALP: alkaline phosphatase; GGT: γ-glutamyltransferase; ALT: alanineaminotransferase; AST: aspartate aminotransferase

Table 32 shows the influence of diet-exposure on blood parameters andliver enzymes within batch. Although the leukocyte counts is within thereference interval from initial to week 3, it was found a verysignificant decrease (p<0.001). After 6 weeks of exposure leukocytecounts was under reference interval (p<0.001) because of a severelymphopenia for all batches (data not shown). Destruction of lymphoidcells could suggest infection agents, but animals showed no other signsof disease. Moreover, neutrophilia was found (data not shown) whichsuggests stress signs. The activity of ALP did not change in any batchduring 6 weeks and the high level ALP activity demonstrated that allbatches had the same amount of stress. AST and GGT remained high in allgroups of animals during 3 weeks (not significant differences; p>0.05)but both decreased significantly to week 6 (p<0.05). Hong Yu et al.,(Hong Yu, VMD; En-dong Bao, PhD; Ru-qian Zhao, PhD; Qiong-xia Lv, VMD.(2007). Effect of transportation stress on heat shock protein 70concentration and mRNA expression in heart and kidney tissues and serumenzyme activities and hormone concentrations of pigs. November 2007,Vol. 68, No. 11, Pages 1145-1150) observed that a severe stress on pigscould manifest as increased serum activities of AST.

Anatomical and Pathological Lesions.

Table 33 shows ZEA contaminated diets produced an increase on vaginaweight (p<0.05) after 3 weeks of exposure. NT and ET-batches had similarvagina weight to ZEA group. Kurtz et al., (Kurtz. H. J., M. E. Naim, G.H. Nelson, C. M. Christensen and C. J. Mirocha. 1969. Histologic changesin the genital tracts of swine fed estrogenic mycotoxin. Am. J. Vet.Res. 30551) observed metaplasia of the epithelium in the cervix andvagina of 6-week-old pre-pubertal gilts fed ZEA (1 mg/day per animal for8 days) which could explain our results. It was also observed that a 20%of the ZEA-treated gilts had follicules >6 mm in size and hypoplasicovary (FIG. 5). Perhaps the estrogenic stimulation of ZEA was so strongthat even the hypoplasic ovaries responded to the ZEA effects. Nofollicules >6 mm in size and hypoplasic ovary were found for the rest ofthe batches. ZEA and NT-treated batches had the highest percentages ofhypoplastic ovaries (p>0.05) (Table 36). It was noticed that hypoplasicand ovulated ovaries were only observed in some gilts of ZEA batch(25%).

After 6 weeks of exposure, the pathological results were significantlydifferent among batches. These differences were (Table 34):

-   -   Oviduct weight: ZEA≧ZEA+NT≈ZEA+ET>CON    -   Weight and size of uterine horns: ZEA=ZEA+NT≧ZEA+ET>CON    -   Cervix weight: ZEA+ET≧ZEA+NT≧ZEA≧CON    -   Vagina weight: ZEA=ZEA+NT>ZEA+ET>CON    -   Vagina size: ZEA=CON=ZEA+NT>ZEA+ET    -   Reproductive organ weight (oviduct-cervix):        ZEA=ZEA+NT≈ZEA+ET>CON

It was also found organ weight differences: there were not significantdifferences among ZEA, ZEA+NT and ZEA+ET butches but, they were overallsignificantly higher than control batch. These results demonstrated theinclusion of ZEA in the diet caused an over-growth reproductive organafter 6 weeks of exposure (p<0.01). Nevertheless, the addition of ET indiet showed the lowest reproductive weight within ZEA treatments. Table34 shows hyperestrogenic symptoms caused by ZEA exposure at week 6.Different reproductive elements enlarged and gained weight when ZEAexposure. In most cases, enlargement and weight gain of ZEA andNT-treated batches were significantly different to control andET-treated gilts. As expected, ZEA treated gilts showed the highestvalues, and control batch the lowest ones. NT and ET provided a partialtoxic sparing effect of ZEA. Furthermore, 20% of the ZEA; 22% of NT and10% of ET-treated gilts had follicules >6 mm in size and hypoplasicovary growth (FIG. 5), but no cases were found in control.

Table 37 showed histopathological lesions of different organs after 6weeks of diet-exposure. All ET-treated gilts had pyelitis (60% moderateand 40% severe) and it was significantly higher (p<0.05) to the othergroups of animals. Liver lesions such as interstitial hepatitis andconnective tissue increment were significantly lower to control batch(p<0.05) which demonstrated ZEA-diet exposure produced a liver toxicitycharacteristics. Neither NT nor ET supplementation minimized thehepatotoxicity. It was also found high percentage of mononuclearcolangiohepatitis cases in gilts of all batches (Table 37), includedcontrol one. Because of high ALP activity during the trial,colangiohepatitis might be the result of a long period of stressexposure.

Table 35 shows the time effect of diet-exposure on reproductive organstructures within batches. A significant reproductive organ weightincrease was found for all batches. An increase of control batchindicated a physiological growth. Observed reproductive organ structuregrowth and enlargement pattern were similar for ZEA and NT-treatedbatches and different to ZEA+ET and control batches. ET-treated andcontrol batches had nearly similar pattern growth during the trial.These data demonstrate that ET overcame ZEA hyperestrogenims symptomsmore efficiently than did NT.

Another observed effect was the number of ovaries having follicules ≦6mm in size. All ovaries recorded after sacrifice were classified by thelargest size of follicle as small/medium (≦6 mm) and large (>6 mm). FIG.3 shows low percentage of ovaries having small follicules to week 3 anda significant increase to week 6, except for ET-treated group. Thepercentage of ovaries having small follicules was not significantlydifferent in the same week (p>0.05). FIG. 4 shows large follicules werefound in ZEA-treated batch and none for the remainders after 3 weeks ofdiet-exposure. Within 6 weeks of exposure, large follicules weredetected in all groups of gilts. The lowest counts were found in controland ET-treated batches. Overall, there were no significant differences(p>0.05). It is likely that any ZEA induced number or size folliculesincrement was masked by the effect of endogenous production, since giltswere approaching puberty. Gilts of this age and weight have aprepubertal ovarial follicular growth rate but it never ends in matureand large follicules (>6 mm) or ovulation process. Follicules >6 mm insize and ovulated ovarian was not expected at the age of experimentalgilts, then estrogenic activity of ZEA could promote theseirregularities and it also explained congestion lesions found inoviducts and ovaries. After 3 weeks of exposure, the ZEA batchregistered 20% of large follicules cases (>6 mm) and none for theremainders (FIG. 5). Within 6 weeks, large follicules were observed inall batches. Control and ET-supplemented batches registered the lowestcases (p<0.05). Ovarian hyperactivity (follicules >6 mm in size andhypoplasic ovary) increased during trial for all batches except forcontrol. ET showed the lowest increment (p<0.05) which suggested that ETalleviates the ovarian hyperactiviy ZEA induces.

TABLE 33 Mean and standard deviation of reproductive organ structuremeasurements. Results obtained after 3 weeks of different diet exposure.3 weeks Control ZEA ZEA + NT ZEA + ET Sig ⁽*⁾ Ovary Weight (g) 0.62 ±0.25 0.25 ± 0.25 0.37 ± 0.25 0.55 ± 0.25 NS Size (cm) 1.55 ± 0.20 1.20 ±0.20 1.18 ± 0.20 1.38 ± 0.20 NS Oviducts: Weight (g) 0.40 ± 0.08 0.44 ±0.08 0.45 ± 0.08 0.38 ± 0.08 NS Size (cm) 14.88 ± 1.55  12.65 ± 1.55 11.85 ± 1.55  13.15 ± 1.55  NS Uterine horns: Weight (g) 8.09 ± 2.4010.50 ± 2.40  10.08 ± 2.40  10.64 ± 2.40  NS Size (cm) 32.45 ± 4.05 32.70 ± 4.05  35.45 ± 4.05  35.90 ± 4.05  NS Cervix Weight (g) 0.40 ±0.14 0.58 ± 0.13 0.44 ± 0.13 0.51 ± 0.13 NS Size (cm) 0.63 ± 0.10 0.70 ±0.09 0.70 ± 0.09 0.90 ± 0.09 NS Vagina Weight (g) 24.34 ± 3.75a 36.13 ±2.37b 38.64 ± 3.06b  33.96 ± 2.37ab * Size (cm) 21.75 ± 1.87  21.70 ±1.18  20.83 ± 1.52  21.80 ± 1.18  NS Weight of reproductive 10.80 ± 3   11.77 ± 2.68  11.34 ± 2.68  12.08 ± 2.68  NS organ (oviducts-cervix; g)⁽*⁾ ^(a-b)Within a row, value with a superscript letter wassignificantly different * (p < 0.05); NS = Not significant (p > 0.05)

TABLE 34 Mean and standard deviation of reproductive organ structuremeasurements. Results obtained after 6 weeks of different diet exposure.6 weeks Control ZEA ZEA + NT ZEA + ET Sig ⁽*⁾ Ovary Weight (g) 1.34 ±0.18 1.23 ± 0.18 1.21 ± 0.19 0.93 ± 0.18 NS Size (cm) 1.99 ± 0.14 1.93 ±0.14 1.85 ± 0.15 1.65 ± 0.14 NS Oviducts: Weight (g)  0.53 ± 0.05a  0.77± 0.05b  0.65 ± 0.06ab  0.67 ± 0.05ab * Size (cm) 14.76 ± 1.10  14.73 ±1.10  16.18 ± 1.15  16.79 ± 1.10  NS Uterine horns: Weight (g) 12.63 ±2.03a 26.07 ± 1.7bc  25.70 ± 1.90bc 20.59 ± 1.79b *** Size (cm) 37.96 ±3.43a  56.53 ± 2.87bc  49.94 ± 3.21bc 47.75 ± 3.02b ** Cervix Weight (g) 0.30 ± 0.11a  0.53 ± 0.10ab  0.62 ± 0.10bc  0.86 ± 0.10c * Size (cm)0.58 ± 0.08 0.50 ± 0.07 0.56 ± 0.07 0.61 ± 0.07 NS Vagina Weight (g)21.91 ± 5.30a 66.34 ± 5.30c 60.03 ± 3.75c 42.82 ± 5.30b * Size (cm) 26.5 ± 2.64b 27.00 ± 2.64b 31.25 ± 1.87b 16.50 ± 2.64a * Weight ofreproductive 14.97 ± 2.27a 28.17 ± 2.12b 28.11 ± 2.12b 23.06 ± 2.00b *organ (oviducts-cervix) ⁽*⁾ ^(a-c)Within a row, value with a superscriptletter was significantly different * (p < 0.05); ** (p < 0.01); *** (p <0.001); NS = Not significant (p > 0.05)

TABLE 35 Time effect of diet exposure on reproductive organ structuresfor different batches Control ZEA ZEA + NT ZEA + ET Ovary Weight (g) *** ** * Size (cm) * ** * * Oviducts: Weight (g) * ** * ** Size (cm) NS **** NS Uterine horns: Weight (g) ** *** *** ** Size (cm) * *** ** *Cervix Weight (g) NS NS NS NS Size (cm) NS NS NS * Vagina Weight (g) NS** * * Size (cm) NS * * NS Weight of reproductive organ * *** ** **(oviducts-cervix; g) ⁽*⁾ Within a row, value with a superscript letterwas significantly different; * (p < 0.05); ** (p < 0.01); *** (p <0.001); NS = Not significant (p > 0.05)

TABLE 36 Histopathology of reproductive organ, kidney and liver after 3weeks of different diet exposure. Results expressed in percentage ofanimals. Histopathology (3 weeks) Control ZEA ZEA + NT ZEA + ET Sig ⁽*⁾Reproductive organ Cervix (slight) 0% 0% 0% 20%  NS Oviduct (slight) 0%0% 20%  0% NS Ovary Hypoplastic 40%  50%  60%  40%  t Preovulatory 0% 0%20%  0% NS Severe preovulatory 0% 0% 0% 0% NS Severehypoplasia-ovulation 0% 25%  0% 0% t Kidneys Multifocal mononuclearinterstitial nephritis Slight 20%  40%  20%  20%  NS Moderate 80%  60% 60%  40%  NS Severe 0% 0% 0% 0% NS Pyelitis Slight 0% 0% 20%  20%  NSModerate 20%  40%  0% 20%  NS Severe 20%  0% 0% 20%  NS Severecomplications 0% 0% 0% 0% NS Pyelonephritis (slight) 0% 0% 0% 0% NSMultifocal pleomorphic interstitial nephritis Slight 0% 0% 0% 0% NSModerate 0% 0% 0% 0% NS Glomerular dilation Slight 0% 0% 0% 0% NS Severecomplications 0% 0% 0% 0% NS Glomerular congestion (slight) 0% 0% 0% 0%NS Tubular fatty 0% 20%  0% 0% NS degeneration (slight) Marrow fibrosis(slight) 0% 0% 0% 0% NS Liver Fatty degeneration 100%  100%  40%  80% NS Hydropic degeneration 80%  60%  80%  40%  NS Central congestionSlight 0% 0% 20%  0% NS Moderate 100%  80%  40%  80%  NS Distortion ofthe lobular architecture Slight 20%  0% 0% 0% NS Moderate 0% 20%  0% 0%NS Septal hepatitis 100%  80%  60%  60%  NS Interstitial hepatitisSlight 100%  80%  40%  80%  NS Moderate 0% 0% 20%  0% NS Mononuclearcholangiohepatitis Slight 80%  100%  100%  80%  NS Moderate 0% 0% 0% 0%NS Severe fribrosis 0% 0% 0% 0% NS Severe complications 0% 0% 0% 0% NSConnective tissue increment Slight 40%  40%  40%  20%  NS Moderate 0% 0%0% 0% NS Capsular fibrosis 0% 0% 0% 0% NS t = tendency; NS = Notsignificant (p > 0.05)

TABLE 37 Histopathology of reproductive organ, kidney and liver after 6weeks of different diet exposure. Results expressed in percentage ofanimals. Histopathology (6 weeks) Control ZEA ZEA + NT ZEA + ET Sig ⁽*⁾Reproductive organ Cervix (slight) 0%  0% 0% 11% NS Oviduct (slight) 0%70% 33%  22% NS Ovary Hypoplastic 10%   0% 0% 10% NS Preovulatory 0% 33%44%  20% NS Severe preovulatory 0%  0% 11%  20% NS Severehypoplasia-ovulation 0% 22% 11%   0% t Kidneys Multifocal mononuclearinterstitial nephritis Slight 60%  20% 44%  10% NS Moderate 30%  40%22%  60% NS Severe 0% 20% 0%  0% NS Severe complications 0%  0% 22%  20%NS Pyelitis Slight  20% a  20% a  33% a  60% b * Moderate  0% a  10% a 33% a  40% b * Severe 0% 20% 0%  0% NS Severe complications 0%  0% 0% 0% NS Pyelonephritis (slight) 0% 10% 0%  0% NS Multifocal pleomorphicinterstitial nephritis Slight 10%   0% 0%  0% NS Moderate 0% 10% 0%  0%NS Glomerular dilation Slight 0% 20% 0%  0% NS Severe complications 10% 10% 0%  0% NS Glomerular congestion (slight) 0% 10% 10%   0% NS Tubularfatty 0% 10% 0% 20% NS degeneration (slight) Marrow fibrosis (slight) 0%10% 11%  40% NS Liver Fatty degeneration 80%  80% 100%  90% NS Hydropicdegeneration 100%  100%  100%  100%  NS Central congestion Slight 90% 70% 67%  40% NS Moderate 0% 30% 33%  60% NS Distortion of the NS lobulararchitecture Slight 40%  20% 56%  70% NS Moderate 0% 10% 0% 10% NSSeptal hepatitis 80%  100%  100%  90% NS Interstitial hepatitis Slight 10% a  80% b  67% b  70% b * Moderate 30%  20% 33%  30% NS Mononuclearcholangiohepatitis Slight  90% b  80% b  56% b  10% b *** Moderate 10% 10% 44%  40% NS Severe fribrosis 0%  0% 0% 50% t Severe complications 0% 0% 0%  0% NS Connective tissue increment Slight  13% a  60% b  64% b 40% b * Moderate 20%   7% 0% 27% NS Capsular fibrosis 10%  30% 11%  20%NS ⁽*^() a-b) Within a row, value with a superscript letter wassignificantly different; NS = Not significant; * (p < 0.05); *** (p <0.01); (p > 0.05)

Conclusions

A dietary concentration of 1 ppm of Zearalenone and 3000 ppm ofadsorbents did not promote worsening or improvement in productivitytraits after 6 weeks of exposure, nor did they impair liver enzymaticfunction or blood parameters. The ET adsorbent was found to be moreeffective than the NT adsorbent to alleviate visual hyperestrogenismeffects (tumefaction-swelling and enlargement of vulva) from week 3 toweek 6. The histological evaluation demonstrated that hyperactive ovary(hypoplasic ovary and large follicules) induced by zearalenone exposurecould be reduced significantly by adding the ET absorbent to the animaldiet, and that the ET-adsorbent promoted normal hypothalamo-hypohysialresponsiveness to estrogen in that E2 plasma concentration remainedconstant (basal levels).

In summary, 1 ppm of ZEA confers multi-organ toxicity in prepubertalgilts after 6 weeks of exposure and ET provides a partial or completetoxic sparing effect of this mycotoxin.

Example 10

The purpose of this method is to provide an analytical tool for the invitro evaluation of mycotoxin detoxifying efficiency. Potentialsequestering agents or “mycotoxin binders” against several mycotoxinscan be evaluated using this method. Adsorption in an acidic environment(pH 3.0), mimicking the pH of the stomach of a monogastric animal anddesorption at pH 6.8 mimicking conditions in the intestine of amonogastric animal are measured in this assay. The net percentage ofmycotoxin detoxifying efficiency is determined as adsorption percentageminus desorption percentage. This “two-phase” in vitro mycotoxinefficiency test is a relatively simple and a direct measure of toxinbinding efficiency measured at varying pH.

Prepare two 0.1M phosphate buffers (pH 3 and pH 6.8) are prepared. Weigh17.799 g+/−0.1 g Na₂HPO₄.2H₂O in a glass beaker. Dissolve in 950 mlde-ionized water. Check pH of the prepared solution and adjust it to thedesired pH (pH 3 and pH 6.8) by adding H₃PO₄ and 1N NaOH. Fill up to 1 Lwith de-ionized water in a volumetric flask.

A stock solution of zearalenone is prepared by dissolving 10 mg ofzearalenone (Sigma or equivalent) in 100 ml acetonitrile (HPLC grade).

Description of Test Procedure Step 1—Adsorption Under Acidic pHConditions (Mimicking Gastric pH)

Prepare a “buffer at pH 3.0 for the toxin stock solution” by adding anappropriate amount of the mycotoxin stock solution to the 0.1 Mphosphate buffer pH 3.0 to yield a suggested final concentration of 300ppb. One should note that this concentration is merely a recommendation.

Add an amount (+/−1 mg) of sequestering agent to a centrifuge tube toobtain a final desired concentration (usual sequestering agentconcentrations to be tested are in the range of 0.1-1.0%). Add an amountof “buffer pH 3.0 toxin stock solution” to obtain the sequestering agentconcentration that needs to be tested and cap the centrifuge tube. Acommon test condition is 0.5% sequestering agent in 10 ml buffer, whichcorresponds to 50 mg sequestering agent in 10 ml buffer.

Standards are treated the same way as the samples with the exceptionthat no sequestering agent is added. Therefore, a centrifuge tube filledwith “buffer at pH 3.0 for the toxin stock solution” runs the wholeprocedure together with the adsorption samples. Afterwards, a standardserial dilution is prepared from this standard by diluting inappropriate buffer. HPLC analyses of these standard dilution series areused to calculate the adsorption results of the samples.

Shake for 1 hour at room temperature on a rocking shaker at a speedcapable to distribute the sequestering agent ‘homogeneously’ in thesolution. Centrifuge the tube to yield a clear supernatant. Add thesupernatant to a HPLC vial.

Dispose the remaining supernatant by decantation and pipette into theprovided waste beaker without removal of the sequestering agent sedimentbefore proceeding to step 2.

Step 2—Desorption Under Near Neutral pH Conditions (Mimicking IntestinalpH)

Add an equal amount (as in step 1) of 0.1 M phosphate buffer pH 6.8without toxin to the residue, cap the centrifuge tube and resuspend theresidue. Usually shaking by hand is sufficient, sometimes shaking on avortex mixer is needed.

Prepare a “buffer at pH 6.8 for the toxin stock solution” by addingappropriate amounts of mycotoxin stock solution to the 0.1 M phosphatebuffer pH 6.8.

Standards are treated the same way as the samples with the exceptionthat no sequestering agent is added. Therefore, a centrifuge tube filledwith “buffer pH 6.8 toxin stock solution” runs the whole proceduretogether with the desorption samples. Afterwards, a standard dilutionseries is prepared from this standard by diluting in appropriate buffer.The HPLC analyses of these standard dilution series are used tocalculate the adsorption/desorption results of the samples.

Shake the tubes for 1 hour at room temperature on a rocking shaker at aspeed capable to distribute the sequestering agent homogeneously in thesolution. Centrifuge the tubes to yield a clear supernatant. Add thesupernatant to a HPLC vial.

HPLC Analysis: HPLC Conditions

Due to the different nature of the samples (buffered samples,interference of compounds in the sequestering agents, etc.),modifications in the HPLC conditions may be required. Since standardsare treated and analysed in the same way as the samples were,calculations are done referring to the corresponding standards(standards pH 3.0 for adsorption samples, standards pH 6.8 fordesorption samples).

HPLC Conditions

The buffering of the samples results sometimes in poor chromatographywhen HPLC conditions of the original internal instructions were applied.Acidification of the original eluent by replacing the water with e.g. 2%glacial acetic acid in water (v/v), was beneficial for thechromatography. Therefore, some slight modifications in original HPLCconditions were introduced to overcome the influence of the buffer onthe chromatogram quality.

The HPLC system used was a Pump-Perkin Elmer Series 200 with a PerkinElmer Series 200 autosampler fitted with 100 μl loop. The column was aLUNA 5 um C18-2, 250×4.6 mm. The UV/Vis detector was a Perkin Elmermodel 200, fluorescence, Perkin Elmer Model 200. The HPLC conditions forzearalenone analyses in 0.1 M phosphate buffer pH 3.0 and 6.8 were: Theeluent was 2% acetic acid in water (v/v)/ acetonitrile 50/50 at a flowrate of 1 ml/min; the column temperature was set at 40° C.; an injectionvolume of 20 μl was used; the excitation wavelength was 274 nm and theemission wavelength was 450 nm.

Calculation

Compare the areas and the retention times of the samples with those ofthe appropriate external standard.

Mycotoxin  binding  efficiency  (%) = Adsorption  (%) − Desorption  (%)${{Adsorption}\mspace{14mu} (\%)} = {\frac{\begin{pmatrix}{{{area}\mspace{14mu} {toxin}\mspace{14mu} {{in}\mspace{14mu}}^{``}{buffer}\mspace{14mu} {pH}\mspace{14mu} 3.0\mspace{14mu} {toxin}\mspace{14mu} {solution}^{''}} -} \\{{area}\mspace{14mu} {toxin}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} {of}\mspace{14mu} {step}\mspace{14mu} 1}\end{pmatrix}}{{area}\mspace{14mu} {toxin}\mspace{14mu} {{in}\mspace{14mu}}^{``}{buffer}\mspace{14mu} {pH}\mspace{14mu} 3.0\mspace{14mu} {toxin}\mspace{14mu} {solution}^{''}} \times 100}$${{Desportion}\mspace{14mu} (\%)} = {\frac{{area}\mspace{14mu} {toxin}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} {of}\mspace{14mu} {step}\mspace{14mu} 2}{{area}\mspace{14mu} {toxin}\mspace{14mu} {{in}\mspace{14mu}}^{``}{buffer}\mspace{14mu} {pH}\mspace{14mu} 6.8\mspace{14mu} {toxin}\mspace{14mu} {solution}^{''}} \times 100}$

Remarks

Döll et al. describe their in vitro test procedure in the article “Invitro studies on the evaluation of mycotoxin detoxifying agents fortheir efficacy on deoxynivalenol and zearalenone” (Archives of animalnutrition, August 2004, Vol. 58 (4), pp. 311-324. In this article theyevaluate the effect of different parameters (pH, time of incubation,amount of buffer added) on the detoxifying capacity of a binder in theirtest. They note that all these parameters have their effect on the testresult. When performing these kinds of in vitro tests, which comprisemany different parameters, one should be aware that relatively smallchanges in one or more parameters could have an effect on the testresult.

A study was performed on the in vitro evaluation of the mycotoxindetoxifying efficiency of potential sequestering humic acid containingsubstances derived from peat against zearalenone. The peat used wasbioAPT™ size 30 fines (small granules) obtained from American PeatTechnologies, Aitkin, Minn. Mineral oil, also known as white oil or oilof a non-vegetable origin, was added to the peat at 3% and 6% by weightto see if it affected binding efficiency. Examples of commercial whiteoil include Citation™ 90 KEM NF Grade Mineral Oil sourced from AVATARCorporation or Duo Prime 90 sourced from Lonza. The peat and peat withoil were compared against Kallsil™, a dry anti-caking aid andnon-nutritive carrier (Kemin Industries, Des Moines, Iowa), vermiculiteand zeolite. Adsorption in an acidic environment (pH 3.0)—mimicking thepH of the stomach of a monogastric animal—and desorption at near neutralpH (pH 6.8)—mimicking pH conditions in the intestine of a monogastricanimal—were measured. The net percentage of mycotoxin detoxifyingefficiency was determined as adsorption percentage minus desorptionpercentage.

Duplicate aliquots of 0.1 M phosphate buffer (adjusted to pH 3.0)containing 300 ppb zearalenone in solution (10 ml) were added to 15 mlscrew cap Falcon polypropylene tubes to which had been added 0.05 gramof each adsorbent. Test tubes were placed on an orbital shaker for 60minutes at room temperature. Each mycotoxin test solution wascentrifuged at 4000 rpm for 10 minutes. The aqueous supernatant wasisolated for mycotoxin analysis (adsorption). The pellet was resuspendedin 0.1 M phosphate buffer pH 6.8. Test tubes were placed again on anorbital shaker for 60 minutes at room temperature and afterwardscentrifuged at 4000 rpm for 10 minutes. The aqueous supernatant wasanalyzed for zearalenone (desorption). The zearalenone detection wasperformed by HPLC analysis. Buffered zearalenone test solutions (pH 3.0or 6.8) without adsorbents were used as standard.

The HPLC analyses were performed on an SP8800 Ternary LC SolventDelivery System with helium degassing (Spectra Physics, USA), a SP 8880autosampler (Spectra Physics, USA) with 20 μl loop, a Chromjetintegrator (Thermo, USA), a Croco Cil™ column heater (Cluzeau Info Labo,France) and an UV-fluorescence detector FP-920 (Jasco, Japan), ChromsepNucleosil 100-5 C18 SS 250*4.6 mm (L*ID) columns (Varian, theNetherlands) or equivalent. The columns were protected with anappropriate guard column. An aliquot of the original bufferedzearalenone test solution was used as the HPLC standard.

A summary of the in vitro mycotoxin adsorption by five samples ispresented in Table 38. The results showed that all the productseffectively adsorbed zearalenone at pH 3.0 (adsorption above the minimalrequired value of 85%), except for zeolite and vermiculite with 3%mineral oil. Notable differences were observed for the desorption at pH6.8. The products vermiculite with 3% oil and peat with oil both wereshown to exceed the maximal value of 10% desorption. The other threesamples met the requirement.

TABLE 38 In vitro binding of zearalenone by humic acid containingsubstances at pH 3.0 and 6.8. In vitro ZEA Binding Adsorption (%)Desorption (%) Efficiency (%) Adsorbent Mean SD Mean SD Mean Kallsil 907 83 Peat/3% Oil 89 21 68 Peat/6% Oil 89 22 67 Peat 85 10 75 Ver/Oil 7913 66 Ver 85 7 78 Zeolite 0 0 0

These results demonstrate that peat binds aflatoxin and zearalenonesubstantially the same as zeolite, thus providing an alternative bindingmaterial that is an organic material instead of a mineral.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art that have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

We claim:
 1. A mycotoxin binder for animal feeds, comprising humicsubstances containing substances derived from peat and an in vitromycotoxin binding efficiency for zearalenone of at least 80% withadsorption of at least 80% at the biological pH of the stomach of amonogastric animal and desorption not greater than 10% at neutral pH. 2.The mycotoxin binder of claim 1, further comprising mineral oil of up to6% by weight of the humic substances.